Targets for regulation of angiogenesis

ABSTRACT

The present invention relates to the identification of polynucleotides and polypeptides having increased expression in tumor blood vessels. The invention further relates to the use of the identified polynucleotides and polypeptides, and inhibitors of the polynucleotides and polypeptides, in the regulation of angiogenesis and the diagnosis and treatment of angiogenesis-related diseases such as cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/945,298, filed Nov. 12, 2010, which is a continuation-in-partapplication of, and claims priority to, International Application No.PCT/US2009/003047; filed on May 15, 2009, which claims the benefit ofU.S. Provisional Application No. 61/053,397, filed May 15, 2008. U.S.application Ser. No. 12/945,398, filed Nov. 12, 2010, also claimspriority to U.S. Provisional Application No. 61/316,068, filed Mar. 22,2010. The entire contents of each of these applications is fullyincorporated herein by reference.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under Grant Nos.CA098034 and CA058223 awarded by the National Institutes of Health andGrant No. W81XWH-04-I-0434 awarded by the Department of Defense. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the identification of polynucleotidesand polypeptides having increased expression in tumor blood vessels. Theinvention further relates to the use of the identified polynucleotidesand polypeptides, and inhibitors of the polynucleotides andpolypeptides, in the regulation of angiogenesis and the diagnosis andtreatment of angiogenesis-related diseases such as cancer.

BACKGROUND OF THE INVENTION

Angiogenesis is the growth of new capillary blood vessels, and is acritical component of solid tumor growth (Folkman, N. Engl. J. Med.285:1182 (1971)). Targeted anti-angiogenic therapy for metastatic breastcancer with bevacizumab, a monoclonal antibody to vascular endothelialgrowth factor (VEGF), has shown efficacy in patients with metastaticbreast cancer (Miller, E2100 Study. Scientific session on monoclonalantibody therapy in breast cancer. Ann. Mtg. Am. Soc. Clin. Oncol. Aug.29, 2005) and validated the approach of anti-angiogenesis therapy forthis disease. Although VEGF is one critical growth factor involved inbreast cancer angiogenesis (Schneider et al., Nat. Clin. Pratt. Oncol.4:181 (2007)), a more detailed understanding of the assortment of genesthat are expressed in breast tumor vessels may facilitate thedevelopment of novel molecularly targeted antiangiogenic agents.

Several studies have established evidence to suggest that blood vesselssupplying tumors express genes not shared by blood vessels that residein normal tissues (Buckanovich et al., J. Clin. Oncol. 25:852 (2007);Madden et al., Am. J. Pathol. 165:601 (2004); Parker et al., Cancer Res.64:7857 (2004); St. Croix et al., Science 289:1197 (2000)). St. Croix etal. used a tissue dissociation and cell immunopurification approach toisolate tumor and normal endothelial cells, and then compared geneexpression patterns of endothelial cells derived from one colorectalcancer and normal colonic mucosa from the same patient (St. Croix etal., Science 289:1197 (2000)). Using serial analysis of gene expression,this analysis identified 46 transcripts, named tumor endothelial markers(TEMs), which were significantly up-regulated in tumor compared withnormal endothelium. Using a similar method, Parker et al. isolatedendothelial cells from two human breast tumors and one normal reductionmammoplasty and identified genes that were differentially expressedcompared to normal breast tissue (Parker et al., Cancer Res. 64:7857(2004)). This study identified 30 breast tumor vascular genes, of whichHEYL and PRL3 were confirmed to be localized in endothelium by in situhybridization. These studies have also shown tumor specific differencesin tumor endothelial markers between colon, breast, and brain tumors.Buckanovich et al. subsequently used laser capture microdissection ofvessel cells from ovarian cancer and normal ovaries and identified 70differentially expressed TEMs (Buckanovich et al., J. Clin. Oncol.25:852 (2007)).

Gene expression studies using DNA microarrays have identified severaldistinct breast cancer subtypes (Perou et al., Nature 406:747 (2000))that differentiate breast cancers into separate groups that differmarkedly in prognosis (Sorlie et al., Proc. Natl. Acad. Sci. USA98:10869 (2001)). The intrinsic subtypes include 2 main subtypes ofestrogen receptor (ER) negative tumors: Basal subtype (ER negative andHer2/neu negative) and Her2/neu subtype (Her2/neu positive and ERnegative); and an ER positive (luminal subtype). Given that TEMs differbetween tumor types, and that breast cancers are molecularlyheterogeneous, it is desirable to determine whether TEMs differ withinthe different molecular subtypes of breast cancer.

The present invention addresses previous shortcomings in the art byproviding novel angiogenesis targets that can be used for diagnostic andtherapeutic methods.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the identification ofpolynucleotides and polypeptides having increased expression in bloodvessels in tumors and the role they play in angiogenesis. The inventionis based further on the use of these polynucleotides and polypeptides,and inhibitors thereof, in the regulation of angiogenesis and thediagnosis and treatment of diseases related to angiogenesis.

Accordingly, as one aspect, the invention provides methods of inhibitingangiogenesis in a cell, comprising decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 in the cell.

In a further aspect, the invention provides methods of inhibitingangiogenesis in a tissue of a subject, comprising decreasing theexpression and/or activity of one or more polypeptides listed in Table 1in the tissue of the subject.

In another aspect, the invention relates to methods of treatingdisorders relating to excessive or undesired angiogenesis in a subject,comprising decreasing the expression and/or activity of one or morepolypeptides listed in Table 1 in the subject.

In another aspect, the invention relates to methods of treating orpreventing cancer in a subject, comprising decreasing the expressionand/or activity of one or more polypeptides listed in Table 1 in thesubject.

The invention further relates to methods of treating or preventingmetastases in a subject, comprising decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 in the subject.

In an additional aspect, the invention relates to methods of reducingtumorigenicity in a subject, comprising decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 in said subject.

In an additional aspect, the invention relates to methods of inhibitingangiogenesis in a tissue of a subject, methods of treating disordersrelating to excessive or undesired angiogenesis in a subject, methods oftreating or preventing cancer in a subject, methods of treating orpreventing metastases in a subject, and/or methods of reducingtumorigenicity in a subject, comprising delivering to the subject acalcineurin or NF-ATc inhibitor, e.g., tacrolimus.

In each of these aspects, the subject may be diagnosed with cancer,e.g., breast cancer. In certain embodiments, the expression and/oractivity of the one or more polypeptides is decreased by decreasing thelevel of a nucleic acid encoding the polypeptide (e.g., with antisenseRNA, microRNA, or siRNA), decreasing the level of the polypeptideitself, or decreasing the activity of the polypeptide (e.g., with anantibody, aptamer, or small molecule that specifically inhibits thepolypeptide itself or a signaling pathway upstream or downstream of thepolypeptide). In one embodiment, the one or more polypeptides isselected from the group consisting of SFRP2, JAK3, and FAP orcombinations thereof. In another embodiment, the one or morepolypeptides does not include SFRP2. In another embodiment, the one ormore polypeptides does not include JAK3. In another embodiment, the oneor more polypeptides does not include FAP.

A further aspect of the invention relates to methods of increasingangiogenesis in a cell, comprising increasing the expression and/oractivity of one or more polypeptides listed in Table 1 in the cell. Incertain embodiments, the expression and/or activity of one or morepolypeptides listed in Table 1 is increased by delivering a nucleic acidencoding the polypeptide or the polypeptide itself to the cell.

Another aspect of the invention relates to methods of increasingangiogenesis in a tissue of a subject, comprising increasing theexpression and/or activity of one or more polypeptides listed in Table 1in the tissue of the subject. In certain embodiments, the expressionand/or activity of one or more polypeptides listed in Table 1 isincreased by delivering a nucleic acid encoding the polypeptide or thepolypeptide itself to the subject.

The invention further relates to methods of diagnosing cancer in asubject, comprising obtaining a sample (e.g., a tissue sample or cells)from the subject and determining the expression and/or activity of oneor more polypeptides listed in Table 1 in the sample, wherein anincrease in expression and/or activity relative to the level ofexpression and/or activity in a control sample is indicative of cancer.In one embodiment, the expression and/or activity of at least 2, 5, 10,or more of the listed polypeptides is determined. In certainembodiments, the expression may be determined by determining the levelof nucleic acid encoding the polypeptide or the polypeptide itself.

An additional aspect of the invention relates to methods of determiningthe angiogenesis potential of a cancer in a subject, comprisingobtaining a sample (e.g., a tissue sample or cells) from the cancer ofthe subject and determining the expression and/or activity of one ormore polypeptides listed in Table 1 in the sample, wherein an increasein expression and/or activity relative to the level of expression and/oractivity in a control sample is indicative of an increased angiogenesispotential of the cancer.

The invention also relates to methods of determining the metastaticpotential of a cancer in a subject, comprising obtaining a sample (e.g.,a tissue sample or cells) from the cancer of the subject and determiningthe expression and/or activity of one or more polypeptides listed inTable 1 in the sample, wherein an increase in expression and/or activityrelative to the level of expression and/or activity in a control sampleis indicative of an increased metastatic potential of the cancer.

Another aspect of the invention relates to methods of monitoring theeffectiveness of a treatment for cancer in a subject, comprisingobtaining a sample (e.g., a tissue sample or cells) from a subject thathas received treatment for cancer, determining the expression and/oractivity of one or more polypeptides listed in Table 1 in the sample,and comparing the level of expression and/or activity to the level ofexpression and/or activity in a control sample, wherein a decrease inthe level of expression and/or activity in the sample relative to thecontrol sample is indicative of the effectiveness of the treatment.

The invention further relates to methods of monitoring the progressionof cancer in a subject, comprising obtaining a sample (e.g., a tissuesample or cells) from a subject that has cancer, determining theexpression and/or activity of one or more polypeptides listed in Table 1in the sample, and comparing the level of expression and/or activity tothe level of expression and/or activity in a control sample, wherein anincrease in the level of expression and/or activity in the samplerelative to the control sample is indicative of progression of thecancer.

The invention also relates to methods of distinguishing among breastcancer subtypes, comprising obtaining a breast cancer sample from asubject, determining the expression and/or activity of one or morepolypeptides listed in Table 1 in the sample, and determining thesubtype of cancer based on the pattern of expression and/or activity. Inone embodiment, the method is used to distinguish between ER negativeand ER positive breast cancers. In another embodiment, the method isused to distinguish between basal, Her2/neu, and luminal subtypes.

The invention further relates to methods of distinguishing between insitu and invasive breast cancers, comprising obtaining a breast cancersample from a subject, determining the expression and/or activity of oneor more polypeptides listed in Table 1 in the sample, and determiningthe type of cancer based on the pattern of expression and/or activity.

Additionally, the invention relates to methods of identifying a compoundthat regulates angiogenesis, comprising determining the expressionand/or activity of one or more polypeptides listed in Table 1 in acell-based assay or a non-cell-based assay in the presence and absenceof a test compound, and selecting a compound that increases or decreasesthe level of expression and/or activity of the one or more polypeptidesrelative to the level in the absence of the compound, as a compound thatregulates angiogenesis.

Another aspect of the invention relates to methods of identifying acompound useful for inhibition of tumor growth or metastasis, comprisingdetermining the expression and/or activity of one or more polypeptideslisted in Table 1 in a cell-based assay or a non-cell-based assay in thepresence and absence of a test compound, and selecting a compound thatincreases or decreases the level of expression and/or activity of theone or more polypeptides relative to the level in the absence of thecompound, as a compound useful for inhibition of tumor growth ormetastasis.

The invention also relates to nucleic acid (e.g., oligonucleotide) orpolypeptide (e.g., antibody) arrays comprising nucleic acids encoding atleast two polypeptides listed in Table 1, e.g., at least 5, 10, 15, 20,or more polypeptides.

The invention further relates to molecules that increase or decrease theexpression and/or activity of a polypeptide listed in Table 1 or anucleic acid encoding the polypeptide. The molecules may be, forexamples, antisense RNA, siRNA, aptamers, antibodies, small molecules,and the like. In one embodiment, the invention relates to pharmaceuticalcompositions comprising the molecules.

In another aspect, the invention relates to kits for assessingangiogenesis, comprising a reagent for determining the expression and/oractivity of one or more polypeptides listed in Table 1.

In a further aspect, the invention relates to kits for diagnosingcancer, comprising a reagent for determining the expression and/oractivity of one or more polypeptides listed in Table 1.

In another aspect, the invention relates to kits for determining themetastatic potential of a cancer, comprising a reagent for determiningthe expression and/or activity of one or more polypeptides listed inTable 1.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows laser capture microdissection of human breast vascularcells before and after microdissection (400×).

FIG. 2 shows RNA integrity analyses. RT-PCR primers for genes of low andhigh abundance levels were used on cDNA from “Whole mount,” which refersto a frozen section of the whole tumor prior to microdissection, and“LCM,” which refers to the sample of vessels microdissected from afrozen section of a human breast tumor. Lane 1, DNA ladder; Lane 2, 3′end of the low expressed ADP ribosylation factor I gene (ARF F1) fromthe “Whole mount” (239 bp); Lane 3, 5′ end of ARF F1 from the “Wholemount” (336 bp); Lane 4, 3′ end of the housekeeping gene GAPDH from the“Whole mount” (540 bp); Lane 5, 5′ end of GAPDH from the “Whole mount”(887 bp); Lane 6, 3′ end of ARF F1 from the microdissected vessel cells;Lane 7, 5′ end of ARF F1 from the microdissected vessel cells; Lane 8,3′ end of GAPDH from the microdissected vessel cells; Lane 9, 5′ end ofGAPDH from the microdissected vessel cells.

FIG. 3 shows gene expression analysis confirming vascular identity.Arrays for LCM vessel cells, endothelial cell lines, and breasttumor-derived cell lines were ordered from left to right. Arrays fromendothelial cells cultured in vitro are labeled:Dermal-microvascular-endothelial-cell, Umbilical-vein-endothelial-cell,Umbilical-vein-endothelial-cells, Aortic-smooth-muscle-cell. Arrays frombreast tumor-derived cell lines in vitro are labeled: T47D-1, T47D-2,MCF7, MDA-MB-365, MDA-MB-453, HCC1937-1, HCC1937-2. The data fordifferent gene sets were identified, and clustered within each relevantcategory, which are in descending order: A) endothelial genes, B) TEMs,C) hematopoietic genes, D) pericyte genes, and E) epithelial genes.

FIG. 4 shows confirmation of vascular origin of vascular marker genes.Pictures were taken at 600× magnification.

FIGS. 5A-5D shows differential protein expression of vascular genesbetween breast tumor vessels and normal breast vessels.

FIG. 6 shows that SFRP2 induces angiogenesis on the chorioallantoicmembrane.

FIG. 7 shows that SFRP2 increases endothelial cell migration in a woundscratch assay.

FIG. 8 shows that SFRP2 induces endothelial tube formation at 8 hours ina concentration-dependent manner.

FIG. 9 shows that SFRP2 induces angiogenesis in the mouse MATRIGEL plugassay.

FIG. 10 shows that SFRP2 inhibits hypoxia-induced apoptosis in MECcells.

FIG. 11 shows gene expression profiling of endothelial cells treatedwith and without SFRP2.

FIG. 12 shows Western blot analysis for nuclear and cytoplasmicβ-catenin expression in mouse endothelial cells treated with SFRP2.

FIG. 13 shows Western blot analyses of nuclear fractions of MEC cellstreated with and without SFRP2 (700 pM) for 1 hour.

FIG. 14 shows that tacrolimus inhibits SFRP2-induced mouse endothelialcell tube formation in vitro.

FIG. 15 shows that tacrolimus reverses SFRP2-induced mouse endothelialcell tube formation in vitro.

FIG. 16 shows that SFRP2 is increased in the SVR angiosarcoma cell linecompared to control mouse endothelial cells.

FIGS. 17A-17D show A) SFRP2 induces tube formation in MEC cells; B)tacrolimus inhibits tube formation in SFRP2-induced MEC cells; C)tacrolimus inhibits tube formation in SVR angiosarcoma cells; and D) SVRtube formation is inhibited by a polyclonal antibody to SFRP2.

FIG. 18 shows that tacrolimus inhibits SFRP2-induced mouse endothelialcell tube formation in 2H11 cells in vitro.

FIG. 19 shows that tacrolimus inhibits VEGF-induced mouse endothelialcell tube formation in 2H11 cells in vitro.

FIGS. 20A-20B show that a polyclonal antibody to SFRP2 inhibits SVR tubeformation in vitro.

FIG. 21 shows that a siRNA to SFRP2 inhibits SVR tube formation invitro.

FIG. 22 shows the inhibitory activity of polyclonal antibodies raisedagainst different epitopes of SFRP2. Sera from mice immunized againstpeptide sequences from SFRP2 (AbA, AbB, AbC, AbD, AbE) and control serawere used at 1:100 dilution. Antibodies to peptide AbA, AbB, AbC, andAbD all inhibited tube formation, however AbB and AbC had the greatestinhibition. N=4 for all groups.

FIGS. 23A-23C show that monoclonal antibodies raised against SFRP2inhibit tube formation. A) Representative control well. B) Angiosarcomacells treated with supernatant from antibody secreting hybridoma showinginhibition of tube formation. C) Branch points from control angiosarcomacells compared with the supernatants from the 8 hybridomas selected forfurther subcloning.

FIG. 24 shows that SFRP2 is overexpressed in serum of cancer patients.Lanes C1-C3 are controls and lanes P1-P6 are breast cancer patients'serum samples (p<0.0001).

FIG. 25 shows that SFRP2 protein is present in the endothelium in a widevariety of tumor types by immunohistochemistry. Paraffin-embeddedsections of human tumors were stained with an antibody to SFRP2.Pictures are taken at 600× magnification.

FIG. 26 shows that tacrolimus inhibits the growth of SVR angiosarcomaxenografts in nude mice. Picture shows representative control mousetumor and representative tacrolimus treated mouse tumor on day 19 oftreatment.

FIG. 27 shows that tacrolimus inhibits the growth rate of MMTV-neutransgenic mouse tumors (n=12 tacrolimus treated, n=9 no treatment,p=0.04).

FIG. 28 shows the ability of Jak3 to promote angiogenesis in vivo usinga chick chorioallantoic membrane (CAM) assay. The graphs showquantitative analysis of vessels surrounding control versus Jak3-treateddisks. The photographs illustrate angiogenesis in vessels surroundingJak3-treated versus control disks.

FIG. 29 shows the migration properties of Jak3 on HCAEC using a scratchwound assay. The graph shows quantitative analysis of the rate of woundclosure in all wells. The photographs illustrate the relative woundclosure of control versus Jak3-treated cells at 28 hours.

FIG. 30 shows the tube formation properties of Jak3 on HCAEC using anendothelial cell tube formation assay. The graph shows quantitativeanalysis of the number of branch points in all wells. The photographsillustrate the relative tube formation in control versus Jak3-treatedcells.

FIG. 31 shows the effect of Jak3 on hypoxia-induced apoptosis in HCAEC.

FIG. 32 shows HCAEC proliferation in the presence of Jak3.

FIG. 33 shows the role of STAT3 activation in Jak3-mediated tubeformation using a small peptide inhibitor of phosphorylated STAT3(P-STAT3). The graph shows quantitative analysis of the number of branchpoints in all wells. The photographs illustrate the relative tubeformation in Jak3-treated versus Jak3+P-STAT3 inhibitor-treated cells.

FIG. 34 shows that a SFRP2 monoclonal antibody inhibits the growth ofSVR angiosarcoma xenografts in nude mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in more detail withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, patentpublications and other references cited herein are incorporated byreference in their entireties for the teachings relevant to the sentenceand/or paragraph in which the reference is presented.

Nucleotide sequences are presented herein by single strand only, in the5′ to 3′ direction, from left to right, unless specifically indicatedotherwise. Nucleotides and amino acids are represented herein in themanner recommended by the IUPAC-IUB Biochemical Nomenclature Commission,or (for amino acids) by either the one-letter code, or the three lettercode, both in accordance with 37 C.F.R. §1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for cloning genes, amplifying and detectingnucleic acids, and the like. Such techniques are known to those skilledin the art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Ausubel et al. CurrentProtocols in Molecular Biology (Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc., New York).

I. Definitions

As used in the description of the invention and the appended claims, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The term “consists essentially of” (and grammatical variants), asapplied to a polynucleotide or polypeptide sequence of this invention,means a polynucleotide or polypeptide that consists of both the recitedsequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5′and/or 3′ or N-terminal and/or C-terminal ends of the recited sequencesuch that the function of the polynucleotide or polypeptide is notmaterially altered. The total of ten or less additional nucleotides oramino acids includes the total number of additional nucleotides or aminoacids on both ends added together. The term “materially altered,” asapplied to polynucleotides of the invention, refers to an increase ordecrease in ability to express the encoded polypeptide of at least about50% or more as compared to the expression level of a polynucleotideconsisting of the recited sequence. The term “materially altered,” asapplied to polypeptides of the invention, refers to an increase ordecrease in angiogenesis-stimulating activity of at least about 50% ormore as compared to the activity of a polypeptide consisting of therecited sequence.

The term “regulate,” “regulates,” or “regulation” refers to enhancement(e.g., an increase) or inhibition (e.g., a decrease) in the specifiedlevel or activity.

The term “enhance” or “increase” refers to an increase in the specifiedparameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold.

The term “inhibit” or “reduce” or grammatical variations thereof as usedherein refers to a decrease or diminishment in the specified level oractivity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%,95% or more. In particular embodiments, the inhibition or reductionresults in little or essentially no detectible activity (at most, aninsignificant amount, e.g., less than about 10% or even 5%).

A “therapeutically effective” amount as used herein is an amount thatprovides some improvement or benefit to the subject. Alternativelystated, a “therapeutically effective” amount is an amount that willprovide some alleviation, mitigation, or decrease in at least oneclinical symptom in the subject (e.g., in the case of cancer, reductionin tumor burden, prevention of further tumor growth, prevention ofmetastasis, or increase in survival time). Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

By the terms “treat,” “treating,” or “treatment of,” it is intended thatthe severity of the subject's condition is reduced or at least partiallyimproved or modified and that some alleviation, mitigation or decreasein at least one clinical symptom is achieved.

The phrase “tumorigenicity” refers primarily to the tumor status of acell or cells (e.g., the extent of neoplastic transformation of a cell,the malignancy of a cell, the propensity for a cell to form a tumorand/or have characteristics of a tumor, or simply the presence orabsence of tumor cells in a patient or tissue/organ), which isreflective of a change of a cell or population of cells from a normal tomalignant state. Tumorigenicity indicates that tumor cells are presentin a sample, and/or that the transformation of cells from normal totumor cells is in progress, as may be confirmed by any standard ofmeasurement of tumor development. The change typically involves cellularproliferation at a rate which is more rapid than the growth observed fornormal cells under the same conditions, and which is typicallycharacterized by one or more of the following traits: continued growtheven after the instigating factor (e.g., carcinogen, virus) is no longerpresent; a lack of structural organization and/or coordination withnormal tissue, and typically, a formation of a mass of tissue, or tumor.A tumor, therefore, is most generally described as a proliferation ofcells (e.g., a neoplasia, a growth, a polyp) resulting from neoplasticgrowth and is most typically a malignant tumor. In the case of aneoplastic transformation, a neoplasia is malignant or is predisposed tobecome malignant. Malignant tumors are typically characterized as beinganaplastic (primitive cellular growth characterized by a lack ofdifferentiation), invasive (moves into and destroys surrounding tissues)and/or metastatic (spreads to other parts of the body).

The phrase “disorder related to excessive or undesired angiogenesis,” asused herein, refers to any disease, disorder, or condition in whichunwanted angiogenesis occurs. Examples of such disorders include,without limitation, cancer, infectious diseases, autoimmune disorders,vascular malformations, DiGeorge syndrome, MIT, cavernous hemangioma,atherosclerosis, transplant arteriopathy, obesity, psoriasis, warts,allergic dermatitis, scar keloids, pyogenic granulomas, blisteringdisease, Kaposi's sarcoma, persistent hyperplastic vitreous syndrome,diabetic retinopathy, retinopathy of prematurity, macular degeneration,choroidal neovascularization, primary pulmonary hypertension, asthma,nasal polyps, inflammatory bowel disease, periodontal disease, ascites,peritoneal adhesions, endometriosis, uterine bleeding, ovarian cysts,ovarian hyperstimulation, ectopic pregnancy, arthritis, synovitis,osteomyelitis, and/or osteophyte formation.

The term “cancer,” as used herein, refers to any benign or malignantabnormal growth of cells. Examples include, without limitation, breastcancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, coloncancer, melanoma, malignant melanoma, ovarian cancer, brain cancer,primary brain carcinoma, head-neck cancer, glioma, glioblastoma, livercancer, bladder cancer, non-small cell lung cancer, head or neckcarcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma,small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicularcarcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma,colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroidcarcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenalcarcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortexcarcinoma, malignant pancreatic insulinoma, malignant carcinoidcarcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia,cervical hyperplasia, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myelogenous leukemia, chronic myelogenousleukemia, chronic granulocytic leukemia, acute granulocytic leukemia,hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma,polycythemia vera, essential thrombocytosis, Hodgkin's disease,non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primarymacroglobulinemia, and retinoblastoma. In some embodiments, the canceris selected from the group of tumor-forming cancers.

The term “breast cancer,” as used herein, refers to a cancer that startsin the cells of the breast of a subject. The term includes invasive andin situ cancers, and encompasses all subtypes of breast cancer,including basal subtype (ER negative and Her2/neu negative), Her2/neusubtype (Her2/neu positive and ER negative); and luminal subtype (ERpositive).

The term “control sample,” as used herein, refers to a tissue or cellsample that is used to compare the level of expression and/or activityof one or more polypeptides listed in Table 1 to the level of expressionand/or activity in a sample of interest. The control sample may be, forexample, from a normal (i.e., non-diseased) portion of the same tissueor cell type in the subject, from a different tissue or cell type in thesubject, from a matched individual, or may be a standard derived fromthe average of measurements taken from a population of subjects. Inanother embodiment, the control sample may be from the disease tissue ofthe subject, e.g., at the time of diagnosis, prior to treatment, orafter a stage of treatment.

As used herein, “nucleic acid,” “nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA andDNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemicallysynthesized) DNA or RNA and chimeras of RNA and DNA. The termpolynucleotide, nucleotide sequence, or nucleic acid refers to a chainof nucleotides without regard to length of the chain. The nucleic acidcan be double-stranded or single-stranded. Where single-stranded, thenucleic acid can be a sense strand or an antisense strand. The nucleicacid can be synthesized using oligonucleotide analogs or derivatives(e.g., inosine or phosphorothioate nucleotides). Such oligonucleotidescan be used, for example, to prepare nucleic acids that have alteredbase-pairing abilities or increased resistance to nucleases. The presentinvention further provides a nucleic acid that is the complement (whichcan be either a full complement or a partial complement) of a nucleicacid, nucleotide sequence, or polynucleotide of this invention.

An “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA)that is not immediately contiguous with nucleotide sequences with whichit is immediately contiguous (one on the 5′ end and one on the 3′ end)in the naturally occurring genome of the organism from which it isderived. Thus, in one embodiment, an isolated nucleic acid includes someor all of the 5′ non-coding (e.g., promoter) sequences that areimmediately contiguous to a coding sequence. The term thereforeincludes, for example, a recombinant DNA that is incorporated into avector, into an autonomously replicating plasmid or virus, or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (e.g., a cDNA or a genomic DNA fragment produced by PCR orrestriction endonuclease treatment), independent of other sequences. Italso includes a recombinant DNA that is part of a hybrid nucleic acidencoding an additional polypeptide or peptide sequence. An isolatedpolynucleotide that includes a gene is not a fragment of a chromosomethat includes such gene, but rather includes the coding region andregulatory regions associated with the gene, but no additional genesnaturally found on the chromosome.

The term “isolated” can refer to a nucleic acid, nucleotide sequence orpolypeptide that is substantially free of cellular material, viralmaterial, and/or culture medium (when produced by recombinant DNAtechniques), or chemical precursors or other chemicals (when chemicallysynthesized). Moreover, an “isolated fragment” is a fragment of anucleic acid, nucleotide sequence or polypeptide that is not naturallyoccurring as a fragment and would not be found in the natural state.“Isolated” does not mean that the preparation is technically pure(homogeneous), but it is sufficiently pure to provide the polypeptide ornucleic acid in a form in which it can be used for the intended purpose.

An isolated cell refers to a cell that is separated from othercomponents with which it is normally associated in its natural state.For example, an isolated cell can be a cell in culture medium and/or acell in a pharmaceutically acceptable carrier of this invention. Thus,an isolated cell can be delivered to and/or introduced into a subject.In some embodiments, an isolated cell can be a cell that is removed froma subject and manipulated as described herein ex vivo and then returnedto the subject.

The term “fragment,” as applied to a polynucleotide, will be understoodto mean a nucleotide sequence of reduced length relative to a referencenucleic acid or nucleotide sequence and comprising, consistingessentially of, and/or consisting of a nucleotide sequence of contiguousnucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99%identical) to the reference nucleic acid or nucleotide sequence. Such anucleic acid fragment according to the invention may be, whereappropriate, included in a larger polynucleotide of which it is aconstituent. In some embodiments, such fragments can comprise, consistessentially of, and/or consist of oligonucleotides having a length of atleast about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive nucleotides of a nucleic acid or nucleotidesequence according to the invention.

The term “fragment,” as applied to a polypeptide, will be understood tomean an amino acid sequence of reduced length relative to a referencepolypeptide or amino acid sequence and comprising, consistingessentially of, and/or consisting of an amino acid sequence ofcontiguous amino acids identical or almost identical (e.g., 90%, 92%,95%, 98%, 99% identical) to the reference polypeptide or amino acidsequence. Such a polypeptide fragment according to the invention may be,where appropriate, included in a larger polypeptide of which it is aconstituent. In some embodiments, such fragments can comprise, consistessentially of, and/or consist of peptides having a length of at leastabout 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, or more consecutive amino acids of a polypeptide or amino acidsequence according to the invention.

A “vector” is any nucleic acid molecule for the cloning of and/ortransfer of a nucleic acid into a cell. A vector may be a replicon towhich another nucleotide sequence may be attached to allow forreplication of the attached nucleotide sequence. A “replicon” can be anygenetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome)that functions as an autonomous unit of nucleic acid replication invivo, i.e., capable of replication under its own control. The term“vector” includes both viral and nonviral (e.g., plasmid) nucleic acidmolecules for introducing a nucleic acid into a cell in vitro, ex vivo,and/or in vivo. A large number of vectors known in the art may be usedto manipulate nucleic acids, incorporate response elements and promotersinto genes, etc. For example, the insertion of the nucleic acidfragments corresponding to response elements and promoters into asuitable vector can be accomplished by ligating the appropriate nucleicacid fragments into a chosen vector that has complementary cohesivetermini. Alternatively, the ends of the nucleic acid molecules may beenzymatically modified or any site may be produced by ligatingnucleotide sequences (linkers) to the nucleic acid termini. Such vectorsmay be engineered to contain sequences encoding selectable markers thatprovide for the selection of cells that contain the vector and/or haveincorporated the nucleic acid of the vector into the cellular genome.Such markers allow identification and/or selection of host cells thatincorporate and express the proteins encoded by the marker. A“recombinant” vector refers to a viral or non-viral vector thatcomprises one or more heterologous nucleotide sequences (i.e.,transgenes), e.g., two, three, four, five or more heterologousnucleotide sequences.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Viral vectorsthat can be used include, but are not limited to, retrovirus,lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus,vaccinia virus, herpes virus, Epstein-Barr virus, and adenovirusvectors. Non-viral vectors include plasmids, liposomes, electricallycharged lipids (cytofectins), nucleic acid-protein complexes, andbiopolymers. In addition to a nucleic acid of interest, a vector mayalso comprise one or more regulatory regions, and/or selectable markersuseful in selecting, measuring, and monitoring nucleic acid transferresults (delivery to specific tissues, duration of expression, etc.).

Vectors may be introduced into the desired cells by methods known in theart, e.g., transfection, electroporation, microinjection, transduction,cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a nucleic acid vectortransporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu etal., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., CanadianPatent Application No. 2,012,311, filed Mar. 15, 1990).

In some embodiments, a polynucleotide of this invention can be deliveredto a cell in vivo by lipofection. Synthetic cationic lipids designed tolimit the difficulties and dangers encountered with liposome-mediatedtransfection can be used to prepare liposomes for in vivo transfectionof a nucleotide sequence of this invention (Felgner et al., Proc. Natl.Acad. Sci. USA 84:7413 (1987); Mackey, et al., Proc. Natl. Acad. Sci.U.S.A. 85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)). Theuse of cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes (Feigner et al., Science 337:387 (1989)). Particularly usefullipid compounds and compositions for transfer of nucleic acids aredescribed in International Patent Publications WO95/18863 andWO96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection tointroduce exogenous nucleotide sequences into specific organs in vivohas certain practical advantages. Molecular targeting of liposomes tospecific cells represents one area of benefit. It is clear thatdirecting transfection to particular cell types would be particularlypreferred in a tissue with cellular heterogeneity, such as pancreas,liver, kidney, and the brain. Lipids may be chemically coupled to othermolecules for the purpose of targeting (Mackey, et al., 1988, supra).Targeted peptides, e.g., hormones or neurotransmitters, and proteinssuch as antibodies, or non-peptide molecules can be coupled to liposomeschemically.

In various embodiments, other molecules can be used for facilitatingdelivery of a nucleic acid in vivo, such as a cationic oligopeptide(e.g., WO95/21931), peptides derived from nucleic acid binding proteins(e.g., WO96/25508), and/or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce a vector in vivo as naked nucleic acid(see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859).Receptor-mediated nucleic acid delivery approaches can also be used(Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem.262:4429 (1987)).

The term “transfection” or “transduction” means the uptake of exogenousor heterologous nucleic acid (RNA and/or DNA) by a cell. A cell has been“transfected” or “transduced” with an exogenous or heterologous nucleicacid when such nucleic acid has been introduced or delivered inside thecell. A cell has been “transformed” by exogenous or heterologous nucleicacid when the transfected or transduced nucleic acid imparts aphenotypic change in the cell and/or a change in an activity or functionof the cell. The transforming nucleic acid can be integrated (covalentlylinked) into chromosomal DNA making up the genome of the cell or it canbe present as a stable plasmid.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and encompass both peptides and proteins, unlessindicated otherwise.

A “fusion protein” is a polypeptide produced when two heterologousnucleotide sequences or fragments thereof coding for two (or more)different polypeptides not found fused together in nature are fusedtogether in the correct translational reading frame. Illustrative fusionpolypeptides include fusions of a polypeptide of the invention (or afragment thereof) to all or a portion of glutathione-S-transferase,maltose-binding protein, or a reporter protein (e.g., Green FluorescentProtein, β-glucuronidase, β-galactosidase, luciferase, etc.),hemagglutinin, c-myc, FLAG epitope, etc.

As used herein, a “functional” polypeptide or “functional fragment” isone that substantially retains at least one biological activity normallyassociated with that polypeptide (e.g., angiogenic activity, proteinbinding, ligand or receptor binding). In particular embodiments, the“functional” polypeptide or “functional fragment” substantially retainsall of the activities possessed by the unmodified peptide. By“substantially retains” biological activity, it is meant that thepolypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%,90%, 95%, 97%, 98%, 99%, or more, of the biological activity of thenative polypeptide (and can even have a higher level of activity thanthe native polypeptide). A “non-functional” polypeptide is one thatexhibits little or essentially no detectable biological activitynormally associated with the polypeptide (e.g., at most, only aninsignificant amount, e.g., less than about 10% or even 5%). Biologicalactivities such as protein binding and angiogenic activity can bemeasured using assays that are well known in the art and as describedherein.

By the term “express” or “expression” of a polynucleotide codingsequence, it is meant that the sequence is transcribed, and optionally,translated. Typically, according to the present invention, expression ofa coding sequence of the invention will result in production of thepolypeptide of the invention. The entire expressed polypeptide orfragment can also function in intact cells without purification.

The term “about,” as used herein when referring to a measurable valuesuch as an amount of polypeptide, dose, time, temperature, enzymaticactivity or other biological activity and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount.

II. Polynucleotides and Polypeptides Upregulated in Tumor Blood VesselCells

The inventors have identified and characterized polypeptides, andpolynucleotides encoding the polypeptides, which are significantlyupregulated in tumor blood vessel cells as compared to non-tumor bloodvessels. Table 1 lists 55 polynucleotides that are upregulated at leastfour-fold in cells associated with breast tumor blood vessels. Each ofthese upregulated polynucleotides and polypeptides represents a usefultarget for the study of angiogenesis, tumor formation, growth andmetastasis. Further, these targets are useful for the diagnosis andtreatment of diseases and disorders related to angiogenesis, e.g.,cancer, ischemia, etc. Additionally, these targets can be used to screenfor agents that can be used to diagnose and treat angiogenesis-relateddiseases and disorders. All information associated with the publicallyavailable identifiers and accession numbers in Table 1, including thenucleic acid sequences of the genes and the amino acid sequences of thepolypeptides encoded thereby, is hereby incorporated by reference in itsentirety.

TABLE 1 Upregulated Genes in Tumor Vessel Cells with Greater than FourFold Change GenBank Fold Gene Symbol Accession No. Gene Name Change NAT1NM_000662 N-acetyltransferase 1 (arylamine N- 17.6 acetyltransferase)DHRS2 NM_005794 Dehydrogenase/reductase (SDR family) 11.9 member 2 IF127NM_005532 Interferon, alpha-inducible protein 27 11.7 S100A8 NM_002964S100A8 S100 calcium binding protein A8 11.7 (calgranulin A) MTL5NM_004923 MTL5 Metallothionein-like 5, testis-specific 10.9 (tesmin) FAPNM_004460 FAP Fibroblast activation protein, alpha 10.7 IFI27 NM_005532Interferon, alpha-inducible protein 27 10.1 UNG2 NM_021147 Uracil-DNAglycosylase 2 9.0 THC1546313 8.9 APXL2 AB075840 Apical protein 2 8.8MGC16121 NM_032762 Hypothetical protein MGC16121 8.7 MMP1 NM_002421Matrix metalloproteinase 1 (interstitial 8.1 collagenase) MMP11NM_005940 Matrix metalloproteinase 11 (stromelysin 3) 8.1 SULF1NM_015170 Sulfatase 1 7.9 SLITRK6 NM_032229 SLIT and NTRK-like family,member 6 7.6 LTB NM_002341 Lymphotoxin beta (TNF superfamily, 7.3 member3) INHBA NM_002192 Inhibin, beta A (activin A, activin AB alpha 7.2polypeptide) THC1598071 6.6 PREX1 NM_020820 Phosphatidylinositol3,4,5-trisphosphate- 6.4 dependent RAC exchanger 1 CHST8 NM_022467Carbohydrate (N-acetylgalactosamine 4-0) 6.4 sulfotransferase 8 SFRP2AF311912 Secreted frizzled-related protein 2 6.3 SMPD3 NM_024703Sphingomyelin phosphodiesterase 3, neutral 6.3 membrane KAZALD1 AF333487Kazal-type serine peptidase inhibitor domain 1 6.2 FGFR3 NM_000142Fibroblast growth factor receptor 3 6.2 SPOCD1 NM_144569 SPOC domaincontaining 1 6.1 IRF7 NM_004030 Interferon regulatory factor 7 5.9COL1A2 NM_000089 Collagen, type I, alpha 2 5.8 CD19 NM_001770 CD19antigen 5.7 BF NM_001710 B-factor, properdin 5.6 SQLE NM_003129 Squaleneepoxidase 5.6 HOXB6 NM_156036 Homeo box B6 5.6 MLPH NM_024101Melanophilin 5.2 DKFZp434E2321 NM_207310 Hypothetical proteinDKFZp434E2321 5.2 HTRA3 NM_053044 HtrA serine peptidase 3 5.1 T3JAMNM_025228 TRAF3-interacting Jun N-terminal kinase 4.9 (JNK)-activatingmodulator ASCL2 NM_005170 Achaete-scute complex-like 2 (Drosophila) 4.9I_960623 4.7 HSPB1 NM_001540 Heat shock 27 kDa protein 1 4.6 COL12A1NM_004370 Collagen, type XII, alpha 1 4.6 HOXB2 NM_002145 Homeo box B24.6 HIG2 NM_013332 Hypoxia-inducible protein 2 4.6 FLJ00332 BC036873FLJ00332 protein 4.6 JAK3 BC028068 Janus kinase 3 (a protein tyrosinekinase, 4.5 leukocyte) S100P NM_005980 S100 calcium binding protein P4.5 RAMP1 NM_005855 Receptor (calcitonin) activity modifying 4.4 protein1 COL5A1 NM_000093 Collagen, type V, alpha 1 4.4 CENPF NM_016343Centromere protein F, 350/400ka (mitosin) 4.3 DOK3 BC004867 Dockingprotein 3 4.2 AA516420 AA516420 4.2 NID2 NM_007361 Nidogen 2(osteonidogen) 4.2 I_1000437 4.1 FGD3 NM_033086 FGD1 family, member 34.1 AK098833 Hypothetical gene supported by AK098833 4.1 AEBP1 NM_001129AE binding protein 1 4.0 A_23_BS21882 4.0

III. Inhibition of Angiogenesis

Accordingly, as one aspect, the invention provides methods of inhibitingangiogenesis in a cell, comprising decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 in the cell.

In a further aspect, the invention provides methods of inhibitingangiogenesis in a tissue of a subject, comprising decreasing theexpression and/or activity of one or more polypeptides listed in Table 1in the tissue of the subject.

In another aspect, the invention relates to methods of treating orpreventing cancer in a subject, comprising decreasing the expressionand/or activity of one or more polypeptides listed in Table 1 in thesubject.

The invention further relates to methods of treating or preventingmetastases in a subject, comprising decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 in the subject.

In an additional aspect, the invention relates to methods of reducingtumorigenicity in a subject, comprising decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 in the subject.

The invention further relates to methods of regulating fertility in afemale subject (e.g., preventing conception or terminating a pregnancy),comprising decreasing the expression and/or activity of one or morepolypeptides listed in Table 1 in the subject (see U.S. Pat. No.6,017,949).

In one embodiment of each of these aspects, the subject may be one thathas been diagnosed with cancer, e.g., breast cancer. In anotherembodiment, the subject may be one that is at risk of developing cancer(e.g., predisposed due to hereditary factors, smoking, viral infection,exposure to chemicals, etc.). In another embodiment, the subject may beone that has been diagnosed with another disease or disorder associatedwith excessive or abnormal angiogenesis, e.g., infectious diseases,autoimmune disorders, vascular malformations, DiGeorge syndrome, HHT,cavernous hemangioma, atherosclerosis, transplant arteriopathy, obesity,psoriasis, warts, allergic dermatitis, scar keloids, pyogenicgranulomas, blistering disease, Kaposi's sarcoma, persistenthyperplastic vitreous syndrome, diabetic retinopathy, retinopathy ofprematurity, choroidal neovascularization, primary pulmonaryhypertension, asthma, nasal polyps, inflammatory bowel disease,periodontal disease, ascites, peritoneal adhesions, endometriosis,uterine bleeding, ovarian cysts, ovarian hyperstimulation, arthritis,synovitis, osteomyelitis, and/or osteophyte formation.

In one embodiment, the expression and/or activity of 2, 3, 4, 5, or moreof the polypeptides listed in Table 1 may be decreased. Any singlepolypeptide or combination of polypeptides on the list may be inhibited.It is further contemplated that any one or more polypeptide listed inTable 1 may be excluded from the methods, e.g., the method may bepracticed with any listed polypeptide except SFRP2. In one embodiment,the polypeptides are selected from the group consisting of SFRP2, JAK3,and FAP, or any combination thereof. In another embodiment, the one ormore polypeptides does not include SFRP2. In another embodiment, the oneor more polypeptides does not include JAK3. In another embodiment, theone or more polypeptides does not include FAP.

In one embodiment of the invention, decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 comprisesdecreasing the level of a nucleic acid (DNA or RNA) encoding thepolypeptide or the level of expression of the polypeptide from thenucleic acid. Numerous methods for reducing the level and/or expressionof polynucleotides in vitro or in vivo are known. For example, thecoding and noncoding nucleotide sequences for the polypeptides listed inTable 1 are known to those of skill in the art and are readily availablein sequence databases such as GenBank. An antisense nucleotide sequenceor nucleic acid encoding an antisense nucleotide sequence can begenerated to any portion thereof in accordance with known techniques.

The term “antisense nucleotide sequence” or “antisense oligonucleotide”as used herein, refers to a nucleotide sequence that is complementary toa specified DNA or RNA sequence. Antisense oligonucleotides and nucleicacids that express the same can be made in accordance with conventionaltechniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No.5,149,797 to Pederson et al. The antisense nucleotide sequence can becomplementary to the entire nucleotide sequence encoding the polypeptideor a portion thereof of at least 10, 20, 40, 50, 75, 100, 150, 200, 300,or 500 contiguous bases and will reduce the level of polypeptideproduction.

Those skilled in the art will appreciate that it is not necessary thatthe antisense nucleotide sequence be fully complementary to the targetsequence as long as the degree of sequence similarity is sufficient forthe antisense nucleotide sequence to hybridize to its target and reduceproduction of the polypeptide. As is known in the art, a higher degreeof sequence similarity is generally required for short antisensenucleotide sequences, whereas a greater degree of mismatched bases willbe tolerated by longer antisense nucleotide sequences.

For example, hybridization of such nucleotide sequences can be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 35-40% formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at37° C.; conditions represented by a wash stringency of 40-45% formamidewith 5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and/orconditions represented by a wash stringency of 50% formamide with5×Denhardt's solution, 0.5% SDS and 1×SSPE at 42° C., respectively) tothe nucleotide sequences specifically disclosed herein. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (ColdSpring Harbor, N.Y., 1989).

In other embodiments, antisense nucleotide sequences of the inventionhave at least about 70%, 80%, 90%, 95%, 97%, 98% or higher sequencesimilarity with the complement of the coding sequences specificallydisclosed herein and will reduce the level of polypeptide production.

In other embodiments, the antisense nucleotide sequence can be directedagainst any coding sequence, the silencing of which results in amodulation of a polypeptide listed in Table 1.

The length of the antisense nucleotide sequence (i.e., the number ofnucleotides therein) is not critical as long as it binds selectively tothe intended location and reduces transcription and/or translation ofthe target sequence, and can be determined in accordance with routineprocedures. In general, the antisense nucleotide sequence will be fromabout eight, ten or twelve nucleotides in length up to about 20, 30, 50,75 or 100 nucleotides, or longer, in length.

An antisense nucleotide sequence can be constructed using chemicalsynthesis and enzymatic ligation reactions by procedures known in theart. For example, an antisense nucleotide sequence can be chemicallysynthesized using naturally occurring nucleotides or various modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleotide sequences, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleotide sequence include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleotide sequencecan be produced using an expression vector into which a nucleic acid hasbeen cloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest).

The antisense nucleotide sequences of the invention further includenucleotide sequences wherein at least one, or all, of theinternucleotide bridging phosphate residues are modified phosphates,such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues can be modified as described. In another non-limiting example,the antisense nucleotide sequence is a nucleotide sequence in which one,or all, of the nucleotides contain a 2′ lower alkyl moiety (e.g., C₁-C₄,linear or branched, saturated or unsaturated alkyl, such as methyl,ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). Forexample, every other one of the nucleotides can be modified asdescribed. See also, Furdon et al., Nucleic Acids Res. 17:9193 (1989);Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al.,Nucleic Acids Res. 18:3537 (1990); Sproat et al., Nucleic Acids Res.17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011(1988); incorporated by reference herein in their entireties for theirteaching of methods of making antisense molecules, including thosecontaining modified nucleotide bases).

Triple helix base-pairing methods can also be employed to inhibitproduction of polypeptides listed in Table 1. Triple helix pairing isbelieved to work by inhibiting the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (e.g., Gee et al., (1994) In: Huber etal., Molecular and Immunologic Approaches, Futura Publishing Co., Mt.Kisco, N.Y.).

Small Interference (si) RNA, also known as RNA interference (RNAi)molecules, provides another approach for modulating the expression ofpolypeptides listed in Table 1. The siRNA can be directed againstpolynucleotide sequences encoding the listed polypeptides or any othersequence that results in modulation of the expression of the listedpolypeptides.

siRNA is a mechanism of post-transcriptional gene silencing in whichdouble-stranded RNA (dsRNA) corresponding to a coding sequence ofinterest is introduced into a cell or an organism, resulting indegradation of the corresponding mRNA. The mechanism by which siRNAachieves gene silencing has been reviewed in Sharp et al., Genes Dev.15:485 (2001); and Hammond et al., Nature Rev. Gen. 2:110 (2001)). ThesiRNA effect persists for multiple cell divisions before gene expressionis regained. siRNA is therefore a powerful method for making targetedknockouts or “knockdowns” at the RNA level. siRNA has proven successfulin human cells, including human embryonic kidney and HeLa cells (see,e.g., Elbashir et al., Nature 411:494 (2001)). In one embodiment,silencing can be induced in mammalian cells by enforcing endogenousexpression of RNA hairpins (see Paddison et al., Proc. Natl. Acad. Sci.USA 99:1443 (2002)). In another embodiment, transfection of small (21-23nt) dsRNA specifically inhibits nucleic acid expression (reviewed inCaplen, Trends Biotechnol. 20:49 (2002)).

siRNA technology utilizes standard molecular biology methods. dsRNAcorresponding to all or a part of a target coding sequence to beinactivated can be produced by standard methods, e.g., by simultaneoustranscription of both strands of a template DNA (corresponding to thetarget sequence) with T7 RNA polymerase. Kits for production of dsRNAfor use in siRNA are available commercially, e.g., from New EnglandBiolabs, Inc. Methods of transfection of dsRNA or plasmids engineered tomake dsRNA are routine in the art.

MicroRNA (miRNA), single stranded RNA molecules of about 21-23nucleotides in length, can be used in a similar fashion to siRNA tomodulate gene expression (see U.S. Pat. No. 7,217,807).

Silencing effects similar to those produced by siRNA have been reportedin mammalian cells with transfection of a mRNA-cDNA hybrid construct(Lin et al., Biochem. Biophys. Res. Commun. 281:639 (2001)), providingyet another strategy for silencing a coding sequence of interest.

The expression of polypeptides listed in Table 1 can also be inhibitedusing ribozymes. Ribozymes are RNA-protein complexes that cleave nucleicacids in a site-specific fashion. Ribozymes have specific catalyticdomains that possess endonuclease activity (Kim et al., Proc. Natl.Acad. Sci. USA 84:8788 (1987); Gerlach et al., Nature 328:802 (1987);Forster and Symons, Cell 49:211 (1987)). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Michel and Westhof, J. Mol. Biol. 216:585(1990); Reinhold-Hurek and Shub, Nature 357:173 (1992)). Thisspecificity has been attributed to the requirement that the substratebind via specific base-pairing interactions to the internal guidesequence (“IGS”) of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, Nature 338:217 (1989)). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., Proc. Natl. Acad.Sci. USA 88:10591 (1991); Sarver et al., Science 247:1222 (1990); Sioudet al., J. Mol. Biol. 223:831 (1992)).

In another embodiment of the invention, decreasing the expression and/oractivity of one or more polypeptides listed in Table 1 comprisesdecreasing the activity of said polypeptide. Polypeptide activity can bemodulated by interaction with an antibody or antibody fragment. Theantibody or antibody fragment can bind to the polypeptide or to anyother polypeptide of interest, as long as the binding between theantibody or the antibody fragment and the target polypeptide results inmodulation of the activity of the listed polypeptide.

The tam “antibody” or “antibodies” as used herein refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibody canbe monoclonal or polyclonal and can be of any species of origin,including (for example) mouse, rat, rabbit, horse, goat, sheep, camel,or human, or can be a chimeric antibody. See, e.g., Walker et al.,Molec. Immunol. 26:403 (1989). The antibodies can be recombinantmonoclonal antibodies produced according to the methods disclosed inU.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibodies canalso be chemically constructed according to the method disclosed in U.S.Pat. No. 4,676,980.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, Fab′, F(ab′)₂, and Fv fragments; domainantibodies, diabodies; vaccibodies, linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. Such fragments can be produced by known techniques. Forexample, F(ab′)₂ fragments can be produced by pepsin digestion of theantibody molecule, and Fab fragments can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments. Alternatively, Fabexpression libraries can be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse et al., Science 254:1275 (1989)).

Antibodies of the invention may be altered or mutated for compatibilitywith species other than the species in which the antibody was produced.For example, antibodies may be humanized or camelized. Humanized formsof non-human (e.g., murine) antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) whichcontain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody)in which residues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Humanized antibodies may also comprise residueswhich are found neither in the recipient antibody nor in the importedCDR or framework sequences. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions (i.e., the sequences between the CDR regions)are those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522 (1986); Riechmann et al.,Nature, 332:323 (1988); and Presta, Curr. Op. Struct. Biol. 2:593(1992)).

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain Humanization canessentially be performed following the method of Winter and co-workers(Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323(1988); Verhoeyen et al., Science 239:1534 (1988)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues (e.g., all of theCDRs or a portion thereof) and possibly some FR residues are substitutedby residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)).The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol. 147:86 (1991)). Similarly, human antibodies can be madeby introducing human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including gene rearrangement, assembly, and antibody repertoire. Thisapproach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in thefollowing scientific publications: Marks et al., Bio/Technology 10:779(1992); Lonberg et al., Nature 368:856 (1994); Morrison, Nature 368:812(1994); Fishwild et al., Nature Biotechnol. 14:845 (1996); Neuberger,Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev.Innunol. 13:65 (1995).

Polyclonal antibodies used to carry out the present invention can beproduced by immunizing a suitable animal (e.g., rabbit, goat, etc.) withan antigen to which a monoclonal antibody to the target binds,collecting immune serum from the animal, and separating the polyclonalantibodies from the immune serum, in accordance with known procedures.

Monoclonal antibodies used to carry out the present invention can beproduced in a hybridoma cell line according to the technique of Kohlerand Milstein, Nature 265:495 (1975). For example, a solution containingthe appropriate antigen can be injected into a mouse and, after asufficient time, the mouse sacrificed and spleen cells obtained. Thespleen cells are then immortalized by fusing them with myeloma cells orwith lymphoma cells, typically in the presence of polyethylene glycol,to produce hybridoma cells. The hybridoma cells are then grown in asuitable medium and the supernatant screened for monoclonal antibodieshaving the desired specificity. Monoclonal Fab fragments can be producedin E. coli by recombinant techniques known to those skilled in the art.See, e.g., Huse, Science 246:1275 (1989).

Antibodies specific to the target polypeptide can also be obtained byphage display techniques known in the art.

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity for the polypeptides of this invention.Numerous protocols for competitive binding or immunoradiometric assaysusing either polyclonal or monoclonal antibodies with establishedspecificity are well known in the art. Such immunoassays typicallyinvolve the measurement of complex formation between an antigen and itsspecific antibody (e.g., antigen/antibody complex formation). Atwo-site, monoclonal-based immunoassay utilizing monoclonal antibodiesreactive to two non-interfering epitopes on the polypeptides or peptidesof this invention can be used as well as a competitive binding assay.

Antibodies can be conjugated to a solid support (e.g., beads, plates,slides or wells formed from materials such as latex or polystyrene) inaccordance with known techniques. Antibodies can likewise be conjugatedto detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescence labels (e.g., fluorescein) in accordance with knowntechniques. Determination of the formation of an antibody/antigencomplex in the methods of this invention can be by detection of, forexample, precipitation, agglutination, flocculation, radioactivity,color development or change, fluorescence, luminescence, etc., as iswell known in the art.

In one embodiment, the antibody is an antibody or a fragment thereof(e.g., a monoclonal antibody) that specifically binds to SFRP2. Theantibody may bind to a specific epitope on SFRP2, e.g., the WNT bindingdomain (about amino acids 30-160) or the NTR domain (about amino acids169-295), that causes inhibition of SFRP2 activity. Suitable epitopesfor raising antibodies include, but are not limited to, sequencescomprising, consisting essentially of, or consisting of amino acids29-40 of human SFRP2 (GQPDFSYRSNC (SEQ ID NO:1)), 85-96 (KQCHPDTKKELC(SEQ ID NO:2)), 119-125 (VQVKDRC (SEQ ID NO:3)) 138-152 (DMLECDRFPQDNDLC(SEQ ID NO:4)), 173-190 (EACKNKNDDDNDIMETLC (SEQ ID NO:5)), 202-220(EITYINRDTKIILETKSKT-Cys (SEQ ID NO:6)), or 270-295(ITSVKRWQKGQREFKRISRSIRKLQC (SEQ ID NO:7)) or a fragment thereof ofthree or more amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more). Inone embodiment, the epitope is amino acids 202-220(EITYINRDTKIILETKSKT-Cys (SEQ ID NO:6)) or a fragment thereof. Inanother embodiment, the epitope is a fragment of the protein from aboutamino acid 156 to about amino acid 295. The amino acid numbering isbased on the GenBank listing for human SFRP2 (accession numberAAH08666), herein incorporated by reference.

In one embodiment, the antibody is a monoclonal antibody produced byhybridoma cell line UNC 68-80 (subclone 80.8.6) (ATCC Deposit No.PTA-11762). In a further embodiment, the antibody is a monoclonalantibody or a fragment thereof that competes for binding to the sameepitope specifically bound by the monoclonal antibody produced byhybridoma cell line UNC 68-80 (ATCC Deposit No. PTA-11762). In anotherembodiment, the antibody is a monoclonal antibody or a fragment thereofthat specifically binds to the same epitope specifically bound by themonoclonal antibody produced by hybridoma cell line UNC 68-80 (ATCCDeposit No. PTA-11762). In certain embodiments, the monoclonal antibodyor a fragment thereof is a chimeric antibody or a humanized antibody. Inadditional embodiments, the chimeric or humanized antibody comprises atleast a portion of the CDRs of the monoclonal antibody produced byhybridoma cell line UNC 68-80 (ATCC Deposit No. PTA-11762). As usedherein, a “portion” of a CDR is defined as one or more of the threeloops from each of the light and heavy chain that make up the CDRs(e.g., from 1-6 of the CDRs) or one or more portions of a loopcomprising, consisting essentially of, or consisting of at least threecontiguous amino acids. For example, the chimeric or humanized antibodymay comprise 1, 2, 3, 4, 5, or 6 CDR loops, portions of 1, 2, 3, 4, 5,or 6 CDR loops, or a mixture thereof.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:13or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a heavy chain variable region comprising anamino acid sequence encoded by the nucleotide sequence of SEQ ID NO:14or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto.

(SEQ ID NO: 13) QVQLQQPGAELVQPGASVMLSCKASGFTFTRYWWHWVRQTPGRGLEWIGRIDPNSGTTRFIEKFKTKATLTVDKPSSTAYMHLSSLTSEDSAVYYCARWGPY YGYAMDYWGPGTSVTVSS(SEQ ID NO: 14) CAGGTCCAATTGCAGCAGCCTGGGGCTGAGCTTGTGCAGCCTGGGGCTTCAGTGATGCTGTCCTGCAAGGCTTCTGGTTTCACCTTCACCAGGTATTGGTGGCACTGGGTGAGGCAGACGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGGTACTACTAGGTTCATTGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACAAACCCTCCAGCACAGCCTACATGCACCTCAGCAGTCTCACATCTGAAGACTCTGCGGTCTATTATTGTGCAAGATGGGGGCCCTACTACGGCTATGCTATGGACTACTGGGGTCCAGGAACCTCAGTCACCGTCT  CCTCA

In one embodiment, the antibody or a fragment thereof comprises a lightchain variable region comprising the amino acid sequence of SEQ ID NO:15or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a light chain variable region comprising anamino acid sequence encoded by the nucleotide sequence of SEQ ID NO:16or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto.

(SEQ ID NO: 15) QIVLTQSPAIMSASPGQKVTITCSASSSVTYMHWYQQKLGSSPKLWIYDTSRLAPGSPARFSGSGSGTSYSLTISSMETEDAASYFCHQWSTYPPTFGTG TKLEIQ(SEQ ID NO: 16) CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGCAGAAAGTCACCATAACCTGCAGTGCCAGTTCAAGTGTTACTTACATGCACTGGTATCAGCAGAAGTTAGGATCCTCCCCCAAACTCTGGATTTATGACACATCCAGACTGGCTCCTGGATCCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGAACCTCTTACTCTCTCACAATCAGCAGCATGGAGACTGAAGATGCTGCCTCTTATTTCTGCCATCAGTGGAGTACTTACCCGCCCACGTTCGGCACGGGG ACAAAATTGGAAATACAA

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:13or a sequence at least 90% identical thereto, e.g., at least 95, 96, 97,98, or 99% identical thereto, or encoded by the nucleotide sequence ofSEQ ID NO:14 or a sequence at least 90% identical thereto, e.g., atleast 95, 96, 97, 98, or 99% identical thereto, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:15 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto, or encoded by the nucleotide sequence of SEQID NO:16 or a sequence at least 90% identical thereto, e.g., at least95, 96, 97, 98, or 99% identical thereto.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising at least one CDR (e.g., 1, 2, or 3) ora portion thereof from the amino acid sequence of SEQ ID NO:13 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a heavy chain variable region comprising atleast one CDR (e.g., 1, 2, or 3) or a portion thereof from an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:14 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto. One of skill in the art understands that theCDRs play an important role in binding specificity and that sequencesubstitutions (e.g., for humanization of a mouse antibody) arepreferably made outside of the CDRs and that minimal changes are madewithin the CDRs. Thus, in some embodiments, sequences that are at least90% identical to the disclosed sequences comprise no changes or only aminimal number of changes to the CDRs.

In one embodiment, the antibody or a fragment thereof comprises a lightchain variable region comprising at least one CDR (e.g., 1, 2, or 3) ora portion thereof from the amino acid sequence of SEQ ID NO:15 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto. In another embodiment, the antibody or afragment thereof comprises a light chain variable region comprising atleast one CDR (e.g., 1, 2, or 3) or a portion thereof from an amino acidsequence encoded by the nucleotide sequence of SEQ ID NO:16 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto.

In one embodiment, the antibody or a fragment thereof comprises a heavychain variable region comprising at least one CDR (e.g., 1, 2, or 3)from the amino acid sequence of SEQ ID NO:13 or a sequence at least 90%identical thereto, e.g., at least 95, 96, 97, 98, or 99% identicalthereto, or encoded by the nucleotide sequence of SEQ ID NO:14 or asequence at least 90% identical thereto, e.g., at least 95, 96, 97, 98,or 99% identical thereto, and a light chain variable region comprisingat least one CDR (e.g., 1, 2, or 3) from the amino acid sequence of SEQID NO:15 or a sequence at least 90% identical thereto, e.g., at least95, 96, 97, 98, or 99% identical thereto, or encoded by the nucleotidesequence of SEQ ID NO:16 or a sequence at least 90% identical thereto,e.g., at least 95, 96, 97, 98, or 99% identical thereto.

In one embodiment, the activity of the of one or more polypeptideslisted in Table 1 is inhibited using aptamers. Recently, smallstructured single-stranded RNAs, also known as RNA aptamers, haveemerged as viable alternatives to small-molecule and antibody-basedtherapy (Que-Gewirth et al., Gene Ther. 14:283 (2007); Ireson et al.,Mol. Cancer Ther. 5:2957 (2006)). RNA aptamers specifically bind targetproteins with high affinity, are quite stable, lack immunogenicity, andelicit biological responses. Aptamers are evolved by means of aniterative selection method called SELEX (systematic evolution of ligandsby exponential enrichment) to specifically recognize and tightly bindtheir targets by means of well-defined complementary three-dimensionalstructures.

RNA aptamers represent a unique emerging class of therapeutic agents(Que-Gewirth et al., Gene Ther. 14:283 (2007); Ireson et al., Mol.Cancer Ther. 5:2957 (2006)). They are relatively short (12-30nucleotide) single-stranded RNA oligonucleotides that assume a stablethree-dimensional shape to tightly and specifically bind selectedprotein targets to elicit a biological response. In contrast toantisense oligonucleotides, RNA aptamers can effectively targetextracellular targets. Like antibodies, aptamers possess bindingaffinities in the low nanomolar to picomolar range. In addition,aptamers are heat stable, lack immunogenicity, and possess minimalinterbatch variability. Chemical modifications, such as amino or fluorosubstitutions at the 2′ position of pyrimidines, may reduce degradationby nucleases. The biodistribution and clearance of aptamers can also bealtered by chemical addition of moieties such as polyethylene glycol andcholesterol. Further, SELEX allows selection from libraries consistingof up to 10¹⁵ ligands to generate high-affinity oligonucleotide ligandsto purified biochemical targets.

In another embodiment, the method of decreasing the activity of apolypeptide listed in Table 1 comprises delivering to a cell or to asubject a compound that decreases the activity of a polypeptide listedin Table 1, the compound administered in an amount effective to modulatethe activity of the polypeptide listed in Table 1. The compound caninteract directly with the polypeptide listed in Table 1 to decrease theactivity of the polypeptide. Alternatively, the compound can interactwith any other polypeptide, nucleic acid or other molecule if suchinteraction results in a decrease of the activity of the polypeptidelisted in Table 1.

The term “compound” as used herein is intended to be interpreted broadlyand encompasses organic and inorganic molecules. Organic compoundsinclude, but are not limited to, small molecules, polypeptides, lipids,carbohydrates, coenzymes, aptamers, and nucleic acid molecules (e.g.,gene delivery vectors, antisense oligonucleotides, siRNA, all asdescribed above).

Polypeptides include, but are not limited to, antibodies (described inmore detail above) and enzymes. Nucleic acids include, but are notlimited to, DNA, RNA and DNA-RNA chimeric molecules. Suitable RNAmolecules include siRNA, antisense RNA molecules and ribozymes (all ofwhich are described in more detail above). The nucleic acid can furtherencode any polypeptide such that administration of the nucleic acid andproduction of the polypeptide results in a decrease of the activity of apolypeptide listed in Table 1.

The compound can further be a compound that is identified by any of thescreening methods described below.

In one embodiment of the invention, the polypeptide listed in Table 1 isSFRP2, which appears to stimulate angiogenesis through activation of thenon-canonical Wnt pathway. The angiogenic activity of SFRP2 can beinhibited by delivering to a subject an inhibitor of this pathway, e.g.,a calcineurin or NF-ATc inhibitor, e.g., an agent that inhibitscalcineurin dephosphorylation of NF-ATc; an agent that inhibits nucleartranslocation of dephosphorylated NF-ATc (agents that block nuclearimport of NF-ATc3 and NF-ATc4; an agent that inhibits DNA binding of anNF-ATc-partner protein binding complex, e.g., through binding to a DNAbinding portion of NF-ATc and/or the partner protein binding region,including agents that inhibit DNA binding by NF-ATc and agents thatprevent the interaction of NF-ATc with their nuclear partner proteins;an agent that reduces the amount of intracellular NF-ATc, e.g., agentsthat inhibit NF-ATc expression (such as antisense or siRNA); or an agentthat enhances the rate of nuclear export by activating GSK3, PKA orother NFAT kinases. Examples of inhibitors that may be used in theinvention include, without limitation, tacrolimus, pimecrolimus,cyclosporin, rapamycin, and the inhibitors disclosed in U.S. Pat. Nos.7,323,439; 7,160,863; 7,084,241; 7,019,028; 6,967,077; 6,875,571;6,780,597; 6,686,450; 6,537,810; 6,399,322; and 5,807,693, each hereinincorporated by reference in its entirety.

The compounds of the present invention can optionally be delivered inconjunction with other therapeutic agents. The additional therapeuticagents can be delivered concurrently with the compounds of theinvention. As used herein, the word “concurrently” means sufficientlyclose in time to produce a combined effect (that is, concurrently can besimultaneously, or it can be two or more events occurring within a shorttime period before or after each other). In one embodiment, thecompounds of the invention are administered in conjunction withanti-cancer agents, such as 1) vinca alkaloids (e.g., vinblastine,vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide);3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g.,L-asparaginase); 5) biological response modifiers (e.g.,interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatinand carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8)substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives(e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocorticalsuppressants (e.g., mitotane (o,p′-DDD) and aminoglutethimide); 11)adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g.,testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g.,flutamide): and 17) gonadotropin-releasing hormone analogs (e.g.,leuprolide). In another embodiment, the compounds of the invention areadministered in conjunction with anti-angiogenesis agents, such asantibodies to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS))and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1),antibodies to alpha-v/beta-3 vascular integrin (e.g., VITAXIN),angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alphaconjugate, cyclophosphamide, combretastatin A4 phosphate,dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin,paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation(Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide,ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate,BMS-582664, CHIR-265, pazopanib, PI-88, vatalanib, everolimus, suramin,sunitinib malate, XL184, ZD6474, ATN-161, cilenigtide, and celecoxib.

IV. Stimulation of Angiogenesis

One aspect of the invention relates to methods of increasingangiogenesis in a cell, comprising increasing the expression and/oractivity of one or more polypeptides listed in Table 1 in the cell.

Another aspect of the invention relates to methods of increasingangiogenesis in a tissue of a subject, comprising increasing theexpression and/or activity of one or more polypeptides listed in Table 1in the tissue of the subject. In one embodiment, the subject is one thathas vascular deficiencies, cardiovascular disease, or would benefit fromthe stimulation of endothelial cell activation and stabilization ofnewly formed microvessels or other vessels, such as in stroke,myocardial infraction, or other types of ischemia, or subjects in needof wound healing, e.g., subjects with ulcers, bed sores, burns, etc.

In one embodiment, increasing the expression and/or activity of one ormore polypeptides listed in Table 1 comprises delivering a nucleic acidencoding the polypeptide or a fragment or homolog thereof to the cell ortissue. In another embodiment, increasing the expression and/or activityof one or more polypeptides listed in Table 1 comprises delivering thepolypeptide itself or a fragment or homolog thereof to the cell ortissue. As used herein, the term “homolog” is used to refer to apolypeptide which differs from a naturally occurring polypeptide byminor modifications to the naturally occurring polypeptide, but whichsignificantly retains a biological activity of the naturally occurringpolypeptide. Minor modifications include, without limitation, changes inone or a few amino acid side chains, changes to one or a few amino acids(including deletions, insertions, and substitutions), changes instereochemistry of one or a few atoms, and minor derivatizations,including, without limitation, methylation, glycosylation,phosphorylation, acetylation, myristoylation, prenylation, palmitation,amidation, and addition of glycosylphosphatidyl inositol. The term“substantially retains,” as used herein, refers to a fragment, homolog,or other variant of a polypeptide that retains at least about 20% of theactivity of the naturally occurring polypeptide (e.g., angiogenicactivity), e.g., about 30%, 40%, 50% or more. Angiogenic activity can bemeasured by, e.g., measuring cell proliferation, angiogenic sprouting,tubule formation, or migration and invasion ability. Other biologicalactivities, depending on the polypeptide, may include enzyme activity,receptor binding, ligand binding, induction of a growth factor, a cellsignal transduction event, etc.

In one embodiment, the method comprises delivering to the subject anisolated polypeptide listed in Table 1. In exemplary embodiments, thepolypeptide comprises, consists essentially of, or consists of thepublicly known amino acid sequence of the polypeptide (disclosed in theGenBank accession numbers in Table 1) or a functional fragment thereof.In another embodiment, the isolated polypeptide comprises, consistsessentially of, or consists of an amino acid sequence that is at least70% identical, e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the publicly known amino acid sequence or a functionalfragment thereof (and polynucleotide sequences encoding the same).

The polypeptide of the invention also include functional portions orfragments (and polynucleotide sequences encoding the same). The lengthof the fragment is not critical as long as it substantially retains thebiological activity of the polypeptide (e.g., angiogenic activity).Illustrative fragments comprise at least about 4, 6, 8, 10, 12, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more contiguous aminoacids of a polypeptide listed in Table 1.

Likewise, those skilled in the art will appreciate that the presentinvention also encompasses fusion polypeptides (and polynucleotidesequences encoding the same) comprising the polypeptide listed in Table1 (or a functional fragment thereof). For example, it may be useful toexpress the polypeptide (or functional fragment) as a fusion proteinthat can be recognized by a commercially available antibody (e.g., FLAGmotifs) or as a fusion protein that can otherwise be more easilypurified (e.g., by addition of a poly-His tail). Additionally, fusionproteins that enhance the stability of the polypeptide may be produced,e.g., fusion proteins comprising maltose binding protein (MBP) orglutathione-S-transferase. As another alternative, the fusion proteincan comprise a reporter molecule. In other embodiments, the fusionprotein can comprise a polypeptide that provides a function or activitythat is the same as or different from the activity of the polypeptide,e.g., a targeting, binding, or enzymatic activity or function.

Likewise, it will be understood that the polypeptides specificallydisclosed herein will typically tolerate substitutions in the amino acidsequence and substantially retain biological activity. To identifypolypeptides of the invention other than those specifically disclosedherein, amino acid substitutions may be based on any characteristicknown in the art, including the relative similarity or differences ofthe amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like.

Amino acid substitutions other than those disclosed herein may beachieved by changing the codons of the DNA sequence (or RNA sequence),according to the following codon table:

TABLE 2 Amino Acid Codons Alanine Ala A GCA GCC GCG GCT Cysteine Cys CTGC TGT Aspartic acid Asp D GAC GAT Glutamic acid Glu E GAA GAGPhenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG GGT Histidine HisH CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys K AAA AAG Leucine LeuL TTA TTG CTA CTC CTG CTT Methionine Met M ATG Asparagine Asn N AAC AATProline Pro P CCA CCC CCG CCT Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGT Serine Ser S AGC ACT TCA TCC TCG TCT ThreonineThr T ACA ACC ACG ACT Valine Val V GTA GTC GTG GTT Tryptophan Trp W TGGTyrosine Tyr Y TAC TAT

In identifying amino acid sequences encoding polypeptides other thanthose specifically disclosed herein, the hydropathic index of aminoacids may be considered. The importance of the hydropathic amino acidindex in conferring interactive biologic function on a protein isgenerally understood in the art (see, Kyte and Doolittle, J. Mol. Biol.157:105 (1982); incorporated herein by reference in its entirety). It isaccepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle, id.),these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

Accordingly, the hydropathic index of the amino acid (or amino acidsequence) may be considered when modifying the polypeptides specificallydisclosed herein.

It is also understood in the art that the substitution of amino acidscan be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(incorporated herein by reference in its entirety) states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±I); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Thus, the hydrophilicity of the amino acid (or amino acid sequence) maybe considered when identifying additional polypeptides beyond thosespecifically disclosed herein.

In embodiments of the invention, the polynucleotide encoding thepolypeptide listed in Table 1 (or functional fragment) will hybridize tothe nucleic acid sequences specifically disclosed herein or fragmentsthereof under standard conditions as known by those skilled in the artand encode a functional polypeptide or functional fragment thereof.

For example, hybridization of such sequences may be carried out underconditions of reduced stringency, medium stringency or even stringentconditions (e.g., conditions represented by a wash stringency of 35-40%formamide with 5×Denhardt's solution, 0.5% SDS and 1×SSPE at 37° C.;conditions represented by a wash stringency of 40-45% formamide with5×Denhardt's solution, 0.5% SDS, and 1×SSPE at 42° C.; and conditionsrepresented by a wash stringency of 50% formamide with 5×Denhardt'ssolution, 0.5% SDS and 1×SSPE at 42° C., respectively) to thepolynucleotide sequences encoding the polypeptides listed in Table 1 orfunctional fragments thereof specifically disclosed herein. See, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (ColdSpring Harbor, N.Y., 1989).

In other embodiments, polynucleotide sequences encoding the polypeptideslisted in Table 1 have at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or higher sequence identity with the publicly known nucleicacid sequences (disclosed in the GenBank accession numbers in Table 1)or functional fragments thereof and encode a functional polypeptide orfunctional fragment thereof.

Further, it will be appreciated by those skilled in the art that therecan be variability in the polynucleotides that encode the polypeptides(and fragments thereof) of the present invention due to the degeneracyof the genetic code. The degeneracy of the genetic code, which allowsdifferent nucleic acid sequences to code for the same polypeptide, iswell known in the literature (See, e.g., Table 2).

Likewise, the polypeptides (and fragments thereof) of the inventioninclude polypeptides that have at least about 70%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or higher amino acid sequence identity with thepublicly known polypeptide sequences.

As is known in the art, a number of different programs can be used toidentify whether a polynucleotide or polypeptide has sequence identityor similarity to a known sequence. Sequence identity or similarity maybe determined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith &Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identityalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Natl.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., Nucl.Acid Res. 12:387 (1984), preferably using the default settings, or byinspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351 (1987); the method is similar to that described by Higgins &Sharp, CABIOS 5:151 (1989).

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., Meth. Enzymol., 266:460 (1996); blast.wustl/edu/blast/README.html.WU-BLAST-2 uses several search parameters, which are preferably set tothe default values. The parameters are dynamic values and areestablished by the program itself depending upon the composition of theparticular sequence and composition of the particular database againstwhich the sequence of interest is being searched; however, the valuesmay be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., Nucleic Acids Res. 25:3389 (1997).

A percentage amino acid sequence identity value is determined by thenumber of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

In a similar manner, percent nucleic acid sequence identity with respectto the coding sequence of the polypeptides disclosed herein is definedas the percentage of nucleotide residues in the candidate sequence thatare identical with the nucleotides in the polynucleotide specificallydisclosed herein.

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the polypeptides specifically disclosed herein,it is understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than a sequence specificallydisclosed herein, will be determined using the number of amino acids inthe shorter sequence, in one embodiment. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0,”which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Those skilled in the art will appreciate that the isolatedpolynucleotides encoding the polypeptides of the invention willtypically be associated with appropriate expression control sequences,e.g., transcription/translation control signals and polyadenylationsignals.

It will further be appreciated that a variety of promoter/enhancerelements can be used depending on the level and tissue-specificexpression desired. The promoter can be constitutive or inducible,depending on the pattern of expression desired. The promoter can benative or foreign and can be a natural or a synthetic sequence. Byforeign, it is intended that the transcriptional initiation region isnot found in the wild-type host into which the transcriptionalinitiation region is introduced. The promoter is chosen so that it willfunction in the target cell(s) of interest.

To illustrate, the polypeptide coding sequence can be operativelyassociated with a cytomegalovirus (CMV) major immediate-early promoter,an albumin promoter, an Elongation Factor 1-α (EF1-α) promoter, a PγKpromoter, a MFG promoter, or a Rous sarcoma virus promoter.

Inducible promoter/enhancer elements include hormone-inducible andmetal-inducible elements, and other promoters regulated by exogenouslysupplied compounds, including without limitation, the zinc-induciblemetallothionein (MT) promoter; the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter; the T7 polymerase promoter system(see WO 98/10088); the ecdysone insect promoter (No et al., Proc. Natl.Acad. Sci. USA 93:3346 (1996)); the tetracycline-repressible system(Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547 (1992)); thetetracycline-inducible system (Gossen et al., Science 268:1766 (1995);see also Harvey et al., Curr. Opin. Chem. Biol. 2:512 (1998)); theRU486-inducible system (Wang et al., Nat. Biotech. 15:239 (1997); Wanget al., Gene Ther., 4:432 (1997)); and the rapamycin-inducible system(Magari et al., J. Clin. Invest. 100:2865 (1997)).

Other tissue-specific promoters or regulatory promoters include, but arenot limited to, promoters that typically confer tissue-specificity inendothelial cells. These include, but are not limited to, promoters forVE-cadherin, PPE-I, PPE-1-3x, TIE-I, TIE-2, Endoglin, von Willebrand,KDR/flk-1, FLT-I, Egr-1, ICAM-2, VCAM-I, PECAM-I, and aorticcarboxypeptidase-like protein (ACLP).

Moreover, specific initiation signals are generally required forefficient translation of inserted polypeptide coding sequences. Thesetranslational control sequences, which can include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

The present invention further provides cells comprising the isolatedpolynucleotides and polypeptides of the invention. The cell may be acultured cell or a cell in vivo, e.g., for use in therapeutic methods,diagnostic methods, screening methods, methods for studying thebiological action of the polypeptides listed in Table 1, in methods ofproducing the polypeptides, or in methods of maintaining or amplifyingthe polynucleotides of the invention, etc. In another embodiment, thecell is an ex vivo cell that has been isolated from a subject. The exvivo cell may be modified and then reintroduced into the subject fordiagnostic or therapeutic purposes.

In particular embodiments, the cell is an untransformed endothelial cellor a cell from a endothelial cell line. Endothelial cells and cell linesinclude, without limitation, HUVEC, HCEC, HGEC, HMEC-1, HUV-ST, ECY304,ECV304, and EA.hy926. In other embodiments, the cell is a pericyte orother cell type associated with blood vessels.

The isolated polynucleotide can be incorporated into an expressionvector. Expression vectors compatible with various host cells are wellknown in the art and contain suitable elements for transcription andtranslation of nucleic acids. Typically, an expression vector containsan “expression cassette,” which includes, in the 5′ to 3′ direction, apromoter, a coding sequence encoding a polypeptide listed in Table 1 orfunctional fragment thereof operatively associated with the promoter,and, optionally, a termination sequence including a stop signal for RNApolymerase and a polyadenylation signal for polyadenylase.

Non-limiting examples of promoters of this invention include CYC1, HIS3,GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO,TPI, and alkaline phosphatase promoters (useful for expression inSaccharomyces); AOX1 promoter (useful for expression in Pichia);β-lactamase, lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trcpromoters (useful for expression in Escherichia coli); light regulated-,seed specific-, pollen specific-, ovary specific-, pathogenesis ordisease related-promoters, cauliflower mosaic virus 35S, CMV 35Sminimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b bindingprotein, ribulose 1,5-bisphosphate carboxylase, shoot-specificpromoters, root specific promoters, chitinase, stress induciblepromoters, rice tungro bacilliform virus, plant super-promoter, potatoleucine aminopeptidase, nitrate reductase, mannopine synthase, nopalinesynthase, ubiquitin, zein protein, and anthocyanin promoters (useful forexpression in plant cells).

Further examples of animal and mammalian promoters known in the artinclude, but are not limited to, the SV40 early (SV40e) promoter region,the promoter contained in the 3′ long terminal repeat (LTR) of Roussarcoma virus (RSV), the promoters of the E1A or major late promoter(MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) earlypromoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter,baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter,phosphoglycerate kinase (PGK) promoter, ubiquitin (Ube) promoter, analbumin promoter, the regulatory sequences of the mousemetallothionein-L promoter and transcriptional control regions, theubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP, and the like), the promoters of therapeutic genes (of theMDR, CFTR or factor VIII type, and the like), pathogenesis and/ordisease-related promoters, and promoters that exhibit tissuespecificity, such as the elastase I gene control region, which is activein pancreatic acinar cells; the insulin gene control region active inpancreatic beta cells, the immunoglobulin gene control region active inlymphoid cells, the mouse mammary tumor virus control region active intesticular, breast, lymphoid and mast cells; the albumin gene promoter,the Apo AI and Apo AII control regions active in liver, thealpha-fetoprotein gene control region active in liver, the alpha1-antitrypsin gene control region active in the liver, the beta-globingene control region active in myeloid cells, the myelin basic proteingene control region active in oligodendrocyte cells in the brain, themyosin light chain-2 gene control region active in skeletal muscle, andthe gonadotropic releasing hormone gene control region active in thehypothalamus, the pyruvate kinase promoter, the villin promoter, thepromoter of the fatty acid binding intestinal protein, the promoter ofsmooth muscle cell α-actin, and the like. In addition, any of theseexpression sequences of this invention can be modified by addition ofenhancer and/or regulatory sequences and the like.

Enhancers that may be used in embodiments of the invention include butare not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer,an elongation factor I (EF1) enhancer, yeast enhancers, viral geneenhancers, and the like.

Termination control regions, i.e., terminator or polyadenylationsequences, may be derived from various genes native to the preferredhosts. In some embodiments of the invention, the termination controlregion may comprise or be derived from a synthetic sequence, a syntheticpolyadenylation signal, an SV40 late polyadenylation signal, an SV40polyadenylation signal, a bovine growth hormone (BGH) polyadenylationsignal, viral terminator sequences, or the like.

It will be apparent to those skilled in the art that any suitable vectorcan be used to deliver the polynucleotide to a cell or subject. Thevector can be delivered to cells in vivo. In other embodiments, thevector can be delivered to cells ex vivo, and then cells containing thevector are delivered to the subject. The choice of delivery vector canbe made based on a number of factors known in the art, including age andspecies of the target host, in vitro versus in vivo delivery, level andpersistence of expression desired, intended purpose (e.g., for therapyor screening), the target cell or organ, route of delivery, size of theisolated polynucleotide, safety concerns, and the like.

Suitable vectors include plasmid vectors, viral vectors (e.g.,retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associatedvirus and other parvoviruses, lentivirus, poxvirus, or herpes simplexvirus), lipid vectors, poly-lysine vectors, synthetic polyamino polymervectors, and the like.

Any viral vector that is known in the art can be used in the presentinvention. Protocols for producing recombinant viral vectors and forusing viral vectors for nucleic acid delivery can be found in Ausubel etal., Current Protocols in Molecular Biology (Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York) and otherstandard laboratory manuals (e.g., Vectors for Gene Therapy. In: CurrentProtocols in Human Genetics. John Wiley and Sons, Inc.: 1997).

Non-viral transfer methods can also be employed. Many non-viral methodsof nucleic acid transfer rely on normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Inparticular embodiments, non-viral nucleic acid delivery systems rely onendocytic pathways for the uptake of the nucleic acid molecule by thetargeted cell. Exemplary nucleic acid delivery systems of this typeinclude liposomal derived systems, poly-lysine conjugates, andartificial viral envelopes.

In particular embodiments, plasmid vectors are used in the practice ofthe present invention. For example, naked plasmids can be introducedinto muscle cells by injection into the tissue. Expression can extendover many months, although the number of positive cells is typically low(Wolff et al., Science 247:247 (1989)). Cationic lipids have beendemonstrated to aid in introduction of nucleic acids into some cells inculture (Feigner and Ringold, Nature 337:387 (1989)). Injection ofcationic lipid plasmid DNA complexes into the circulation of mice hasbeen shown to result in expression of the DNA in lung (Brigham et al.,Am. J. Med. Sci. 298:278 (1989)). One advantage of plasmid DNA is thatit can be introduced into non-replicating cells.

In a representative embodiment, a nucleic acid molecule (e.g., aplasmid) can be entrapped in a lipid particle bearing positive chargeson its surface and, optionally, tagged with antibodies against cellsurface antigens of the target tissue (Mizuno et al., No Shinkei Geka20:547 (1992); PCT publication WO 91/06309; Japanese patent application1047381; and European patent publication EP-A-43075).

Liposomes that consist of amphiphilic cationic molecules are useful asnon-viral vectors for nucleic acid delivery in vitro and in vivo(reviewed in Crystal, Science 270:404 (1995); Blaese et al., Cancer GeneTher. 2:291 (1995); Behr et al., Bioconjugate Chem. 5:382 (1994); Remyet al., Bioconjugate Chem. 5:647 (1994); and Gao et al., Gene Therapy2:710 (1995)). The positively charged liposomes are believed to complexwith negatively charged nucleic acids via electrostatic interactions toform lipid:nucleic acid complexes. The lipid:nucleic acid complexes haveseveral advantages as nucleic acid transfer vectors. Unlike viralvectors, the lipid:nucleic acid complexes can be used to transferexpression cassettes of essentially unlimited size. Since the complexeslack proteins, they can evoke fewer immunogenic and inflammatoryresponses. Moreover, they cannot replicate or recombine to form aninfectious agent and have low integration frequency. A number ofpublications have demonstrated that amphiphilic cationic lipids canmediate nucleic acid delivery in vivo and in vitro (Feigner et al.,Proc. Natl. Acad. Sci. USA 84:7413 (1987); Loeffler et al., Meth.Enzymol. 217:599 (1993); Feigner et al., J. Biol. Chem. 269:2550(1994)).

Several groups have reported the use of amphiphilic cationiclipid:nucleic acid complexes for in vivo transfection both in animalsand in humans (reviewed in Gao et al., Gene Therapy 2:710 (1995); Zhu etal., Science 261:209 (1993); and Thierry et al., Proc. Natl. Acad. Sci.USA 92:9742 (1995)). U.S. Pat. No. 6,410,049 describes a method ofpreparing cationic lipid:nucleic acid complexes that have a prolongedshelf life.

Expression vectors can be designed for expression of polypeptides inprokaryotic or eukaryotic cells. For example, polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., thebaculovirus expression system), yeast cells, plant cells or mammaliancells. Some suitable host cells are discussed further in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Examples of bacterial vectors include pQE70,pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK,pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR540, and pRIT5 (Pharmacia). Examples of vectors forexpression in the yeast S. cerevisiae include pYepSecl (Baldari et al.,EMBO J. 6:229 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933 (1982)),pJRY88 (Schultz et al., Gene 54:113 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.). Baculovirus vectors available forexpression of nucleic acids to produce proteins in cultured insect cells(e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell.Biol. 3:2156 (1983)) and the pVL series (Lucklow and Summers Virology170:31 (1989)).

Examples of mammalian expression vectors include pWLNEO, pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed,Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187(1987)). When used in mammalian cells, the expression vector's controlfunctions are often provided by viral regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus and Simian Virus 40.

Viral vectors have been used in a wide variety of gene deliveryapplications in cells, as well as living animal subjects. Viral vectorsthat can be used include, but are not limited to, retrovirus,lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus,vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus,geminivirus, and caulimovirus vectors. Non-viral vectors includeplasmids, liposomes, electrically charged lipids (cytofectins), nucleicacid-protein complexes, and biopolymers. In addition to a nucleic acidof interest, a vector may also comprise one or more regulatory regions,and/or selectable markers useful in selecting, measuring, and monitoringnucleic acid transfer results (delivery to specific tissues, duration ofexpression, etc.).

In addition to the regulatory control sequences discussed above, therecombinant expression vector can contain additional nucleotidesequences. For example, the recombinant expression vector can encode aselectable marker gene to identify host cells that have incorporated thevector.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acids (e.g.,DNA and RNA) into a host cell, including calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, electroporation, microinjection, DNA-loaded liposomes,LIPOFECTAMINE DNA complexes, cell sonication, gene bombardment usinghigh velocity microprojectiles, and viral-mediated transfection.Suitable methods for transforming or transfecting host cells can befound in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed.(Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

If stable integration is desired, often only a small fraction of cells(in particular, mammalian cells) integrate the foreign DNA into theirgenome. In order to identify and select integrants, a nucleic acid thatencodes a selectable marker (e.g., resistance to antibiotics) can beintroduced into the host cells along with the nucleic acid of interest.Preferred selectable markers include those that confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acids encodinga selectable marker can be introduced into a host cell on the samevector as that comprising the nucleic acid of interest or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

Polypeptides and fragments of the invention can be modified for in vivouse by the addition, at the amino- and/or carboxyl-terminal ends, of ablocking agent to facilitate survival of the relevant polypeptide invivo. This can be useful in those situations in which the peptidetermini tend to be degraded by proteases prior to cellular uptake. Suchblocking agents can include, without limitation, additional related orunrelated peptide sequences that can be attached to the amino and/orcarboxyl terminal residues of the peptide to be administered. This canbe done either chemically during the synthesis of the peptide or byrecombinant DNA technology by methods familiar to artisans of averageskill. Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

Another embodiment of the invention relates to homologs of thepolypeptides of the invention that are peptidomimetic compounds that aredesigned based upon the amino acid sequences of the functionalpolypeptide fragments. Peptidomimetic compounds are synthetic compoundshaving a three-dimensional conformation (i.e., a “peptide motif”) thatis substantially the same as the three-dimensional conformation of aselected-peptide. The peptide motif provides the peptidomimetic compoundwith the ability to enhance angiogenesis in a manner qualitativelyidentical to that of the functional fragment from which thepeptidomimetic was derived. Peptidomimetic compounds can have additionalcharacteristics that enhance their therapeutic utility, such asincreased cell permeability and prolonged biological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbon A,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

In one embodiment, the polynucleotides, polypeptides, or homologsthereof of the invention are administered directly to the subject.Generally, the compounds of the invention will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous infusion, or injectedsubcutaneously, intramuscularly, intrathecally, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily. They are preferably delivereddirectly to the site of the disease or disorder, such as tumor cells,e.g., to a tumor or a tumor bed following surgical excision of thetumor, in order to kill any remaining tumor cells. The dosage requireddepends on the choice of the route of administration; the nature of theformulation; the nature of the patient's illness; the subject's size,weight, surface area, age, and sex; other drugs being administered; andthe judgment of the attending physician. Suitable dosages are in therange of 0.01-100.0 μg/kg. Wide variations in the needed dosage are tobe expected in view of the variety of polypeptides and fragmentsavailable and the differing efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection.Variations, in these dosage levels can be adjusted using standardempirical routines for optimization as is well understood in the art.Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-,10-; 20-, 50-, 100-, 150-, or more fold). Encapsulation of thepolypeptide in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

According to certain embodiments, the polynucleotides or vectors can betargeted to specific cells or tissues in vivo. Targeting deliveryvehicles, including liposomes and viral vector systems are known in theart. For example, a liposome can be directed to a particular target cellor tissue by using a targeting agent, such as an antibody, solublereceptor or ligand, incorporated with the liposome, to target aparticular cell or tissue to which the targeting molecule can bind.Targeting liposomes are described, for example, in Ho et al.,Biochemistry 25:5500 (1986); Ho et al., J. Biol. Chem. 262:13979 (1987);Ho et al., J. Biol. Chem. 262:13973 (1987); and U.S. Pat. No. 4,957,735to Huang et al., each of which is incorporated herein by reference inits entirety). Enveloped viral vectors can be modified to deliver anucleic acid molecule to a target cell by modifying or substituting anenvelope protein such that the virus infects a specific cell type. Inadenoviral vectors, the gene encoding the attachment fibers can bemodified to encode a protein domain that binds to a cell-specificreceptor. Herpesvirus vectors naturally target the cells of the centraland peripheral nervous system. Alternatively, the route ofadministration can be used to target a specific cell or tissue. Forexample, intracoronary administration of an adenoviral vector has beenshown to be effective for the delivery of a gene cardiac myocytes(Maurice et al., J. Clin. Invest. 104:21 (1999)). Intravenous deliveryof cholesterol-containing cationic liposomes has been shown topreferentially target pulmonary tissues (Liu et al., Nature Biotechnol.15:167 (1997)), and effectively mediate transfer and expression of genesin vivo. Other examples of successful targeted in vivo delivery ofnucleic acid molecules are known in the art. Finally, a recombinantnucleic acid molecule can be selectively (i.e., preferentially,substantially exclusively) expressed in a target cell by selecting atranscription control sequence, and preferably, a promoter, which isselectively induced in the target cell and remains substantiallyinactive in non-target cells.

V. Diagnosis and Monitoring of Angiogenesis-Related Diseases

The identification of polynucleotides and polypeptides that areupregulated in tumor blood vessels provides targets to be used fordetection of angiogenesis and diagnosis of angiogenesis-related diseasesand disorders.

One aspect of the invention relates to methods of detecting angiogenesisin a tissue of a subject, comprising obtaining a sample from the tissueand determining the expression and/or activity of one or morepolypeptides listed in Table 1 in the sample, wherein an increase inexpression and/or activity relative to the level of expression and/oractivity in a control sample is indicative of angiogenesis. In oneembodiment, the tissue is diseased tissue such as cancer tissue, e.g.,breast cancer tissue. In another embodiment, the tissue is not diseasedtissue.

Another aspect of the invention relates to methods of diagnosing cancerin a subject, comprising obtaining a tissue sample from the subject anddetermining the expression and/or activity of one or more polypeptideslisted in Table 1 in the sample, wherein an increase in expressionand/or activity relative to the level of expression and/or activity in acontrol sample is indicative of cancer.

A further aspect of the invention relates to methods of determining theangiogenesis potential of a tissue in a subject, comprising obtaining asample from the tissue of the subject and determining the expressionand/or activity of one or more polypeptides listed in Table 1 in thesample, wherein an increase in expression and/or activity relative tothe level of expression and/or activity in a control sample isindicative of an increased angiogenesis potential of said tissue.

Another aspect of the invention relates to methods of determining themetastatic potential of a cancer in a subject, comprising obtaining atissue sample from the cancer of the subject and determining theexpression and/or activity of one or more polypeptides listed in Table 1in the sample, wherein an increase in expression and/or activityrelative to the level of expression and/or activity in a control sampleis indicative of an increased metastatic potential of said cancer.

In each of these aspects, the expression and/or activity of more thanone polypeptide listed in Table 1 may be determined, e.g., 2, 3, 4, 5,10, 15, 20, 25, or more polypeptides. In one embodiment, said one ormore polypeptides is selected from the group consisting of SFRP2, JAK3and FAP, or combinations thereof. In another embodiment, the one or morepolypeptides does not include SFRP2. In another embodiment, the one ormore polypeptides does not include JAK3. In another embodiment, the oneor more polypeptides does not include FAP. The tissue sample may beobtained by any method known in the art, such as surgery, biopsy,lavage, aspiration, etc. The sample may be a bodily fluid, e.g., blood,serum, plasma, saliva, urine, cerebrospinal fluid, perspiration, etc.The control sample may be from a normal (i.e., non-diseased) portion ofthe same tissue or cell type in the subject, from a different tissue orcell type in the subject, from a matched individual, or may be astandard derived from the average of measurements taken from apopulation of subjects. In one embodiment, the tissue sample is isolatedblood vessels or isolated endothelial cells. Blood vessels can beisolated by any means known in the art and as described herein.Endothelial cells can be isolated by any means known in the art, e.g.,cell sorting, immunoprecipitation, etc.

In one embodiment, the subject has cancer, e.g., breast cancer.

In one embodiment, determining the expression and/or activity of one ormore polypeptides listed in Table 1 comprises determining the level of anucleic acid encoding said one or more polypeptides. Determining thelevel of a nucleic acid can be carried out by any means known in the artand as described herein, such as Northern blots, dot blots, PCR, RT-PCR,quantitative PCR, sequence analysis, gene microarray analysis, in situhybridization, and detection of a reporter gene. Assays for expressionand/or activity can be carried out automatically or partiallyautomatically in a machine or apparatus designed to perform such assays,e.g., using computer-assisted methods. The results of the assays can bestored in a computer database and analyzed to produce diagnosticresults. In some embodiments, the diagnostic data can be analyzed, e.g.,by comparing intra-patient results over time or before and aftertreatment or comparing inter-patient results to determine baselineand/or abnormal values in a population.

In another embodiment, determining the expression and/or activity of oneor more polypeptides listed in Table 1 comprises determining the levelof said one or more polypeptides. Determining the level of a polypeptidecan be carried out by any means known in the art and as describedherein, such as Western blots, immunoblots, immunoprecipitation,immunohistochemistry, immunofluorescence, enzyme-linked immunosorbantassays, and radioimmunoassays.

In a further embodiment, determining the expression and/or activity ofone or more polypeptides listed in Table 1 comprises determining theactivity of said one or more polypeptides. The activity may be anyactivity associated with the polypeptide, including, without limitation,angiogenic activity, enzyme activity, protein interaction, receptorbinding, ligand binding, induction of a growth factor, a cell signaltransduction event, etc.

The invention also relates to methods of distinguishing among breastcancer subtypes, comprising obtaining a breast cancer sample from asubject, determining the expression and/or activity of one or morepolypeptides listed in Table 1 in the sample, and determining thesubtype of cancer based on the pattern of expression and/or activity. Inone embodiment, the method is used to distinguish between ER negativeand ER positive breast cancers. In another embodiment, the method isused to distinguish between basal, Her2/neu, and luminal subtypes.

The invention further relates to methods of distinguishing between insitu and invasive breast cancers, comprising obtaining a breast cancersample from a subject, determining the expression and/or activity of oneor more polypeptides listed in Table 1 in the sample, and determiningthe type of cancer based on the pattern of expression and/or activity.

One aspect of the invention relates to the use of the identified markersof angiogenesis to monitor the regulation of angiogenesis due to diseaseor treatment of the disease. In one aspect, the invention relates tomethods of monitoring the effectiveness of a treatment for cancer in asubject, comprising obtaining a sample from a subject that has receivedtreatment for cancer, determining the expression and/or activity of oneor more polypeptides listed in Table 1 in the sample, and comparing thelevel of expression and/or activity to the level of expression and/oractivity in a control sample, wherein a decrease in the level ofexpression and/or activity in the sample relative to the control sampleis indicative of the effectiveness of the treatment.

Another aspect of the invention relates to methods of monitoring theprogression of cancer in a subject, comprising obtaining a sample from asubject that has cancer, determining the expression and/or activity ofone or more polypeptides listed in Table 1 in the sample, and comparingthe level of expression and/or activity to the level of expressionand/or activity in a control sample, wherein an increase in the level ofexpression and/or activity in the sample relative to the control sampleis indicative of progression of the cancer.

The control sample may be from a normal (i.e., non-diseased) portion ofthe same tissue or cell type in the subject, from a different tissue orcell type in the subject, from a matched individual, or may be astandard derived from the average of measurements taken from apopulation of subjects. In another embodiment, the control sample may befrom the disease tissue of the subject, e.g., at the time of diagnosis,prior to treatment, or after a stage of treatment.

In each of these aspects, a baseline level of expression and/or activitymay be determined upon the initial diagnosis of cancer or prior to thefirst treatment. After a baseline is established, the expression and/oractivity of the one or more polypeptides may be determined repeatedly,e.g., on a regular schedule (e.g., once every 2, 3, 4, 5, or 6 days, 1,2, 3, or 4 weeks, or more) or as desired (e.g., after each therapeutictreatment). Expression and/or activity may be determined as describedabove, and may be at the nucleic acid or polypeptide level. Theinformation obtained from the monitoring may be used to modify thetreatment the subject is receiving.

One aspect of the invention relates to kits useful for carrying out themethods of the invention. One embodiment relates to kits for assessingangiogenesis, comprising a reagent for determining the expression and/oractivity of one or more polypeptides listed in Table 1. Anotherembodiment relates to kits for diagnosing cancer, comprising a reagentfor determining the expression and/or activity of one or morepolypeptides listed in Table 1. In each embodiment, the kits may containreagents for determining the expression and/or activity of 2, 3, 4, 5,10, 15, 20, 25, or more polypeptides listed in Table 1. The reagents maybe nucleic acids (e.g., an oligonucleotide that specifically hybridizesto a nucleic acid encoding a polypeptide listed in Table 1 and can beused as a hybridization probe or an amplification primer), antibodies(e.g., one the specifically binds to a polypeptide listed in Table 1),or other agents that specifically recognize the polynucleotides orpolypeptides of the invention.

The reagents can be conjugated to a detectable tag or detectable label.Such a tag can be any suitable tag which allows for detection of thereagents and includes, but is not limited to, any composition or labeldetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include biotin for staining with labeled streptavidinconjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein, and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

In addition, the reagents can be immobilized on a substrate. Such asubstrate can include any suitable substrate for immobilization of adetection reagent such as would be used in any of the previouslydescribed methods of detection. Briefly, a substrate suitable forimmobilization of a detection reagent includes any solid support, suchas any solid organic, biopolymer or inorganic support that can form abond with the detection reagent without significantly effecting theactivity and/or ability of the detection reagent to detect the desiredtarget molecule. Exemplary organic solid supports include polymers suchas polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers(e.g., polyacrylamide), stabilized intact whole cells, and stabilizedcrude whole cell/membrane homogenates. Exemplary biopolymer supportsinclude cellulose, polydextrans (e.g., Sephadex®), agarose, collagen andchitin. Exemplary inorganic supports include glass beads (porous andnonporous), stainless steel, metal oxides (e.g., porous ceramics such asZrO₂, TiO₂, Al₂O₃, and NiO) and sand.

The kits may further comprise other components useful for detectingexpression or activity, e.g., buffers, cells, culture medium, enzymes,labeling reagents, containers, etc.

In one embodiment, the kit comprises an array of reagents fordetermining expression and/or activity. The array can comprise asubstrate having a plurality of addresses. At least one address of theplurality includes a capture probe that binds specifically to apolynucleotide or polypeptide of the invention. The array may comprisecapture probes corresponding to 5, 10, 15, 20, 25, or more of thepolypeptides listed in Table 1. The array can have a density of atleast, or less than, 10, 20 50, 100, 200, 500, 700, 1,000, 2,000, 5,000or 10,000 or more addresses/cm², and ranges between. The substrate canbe a two-dimensional substrate such as a glass slide, a wafer (e.g.,silica or plastic), a mass spectroscopy plate, or a three-dimensionalsubstrate such as a gel pad. Addresses in addition to addresses of theplurality can be disposed on the array.

In one embodiment, at least one address of the plurality includes anucleic acid capture probe that hybridizes specifically to apolynucleotide of the invention, e.g., the sense or anti-sense strand.Each address of the subset can include a capture probe that hybridizesto a different region of a polynucleotide. An array can be generated byany of a variety of methods. Appropriate methods include, e.g.,photolithographic methods (e.g., U.S. Pat. Nos. 5,143,854; 5,510,270;and 5,527,681), mechanical methods (e.g., directed-flow methods asdescribed in U.S. Pat. No. 5,384,261), pin-based methods (e.g., asdescribed in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g.,as described in PCT US/93/04145).

In another embodiment, at least one address of the plurality includes apolypeptide capture probe that binds specifically to a polypeptide ofthe invention or fragment thereof. The polypeptide capture probe can bea naturally-occurring interaction partner of a polypeptide listed inTable 1, e.g., a where the polypeptide is a receptor or a receptor wherethe polypeptide is ligand. In one embodiment, the polypeptide is anantibody, e.g., an antibody specific for a polypeptide listed in Table1, such as a polyclonal antibody, a monoclonal antibody, or asingle-chain antibody.

VI. Screening Assays and Animal Models

The identification of polynucleotides and polypeptides that areupregulated in tumor blood vessels provides targets that can be used toscreen for agents that regulate angiogenesis as well as models forstudying the process of angiogenesis in vitro or in animals.

One aspect of the invention relates to methods of identifying a compoundthat regulates angiogenesis, comprising determining the expressionand/or activity of one or more polypeptides listed in Table 1 in thepresence and absence of a test compound, and selecting a compound thatincreases or decreases the level of expression and/or activity of theone or more polypeptides relative to the level in the absence of thecompound, as a compound that regulates angiogenesis.

Another aspect of the invention relates to methods of identifying acompound useful for inhibition of tumor growth or metastasis, comprisingdetermining the expression and/or activity of one or more polypeptideslisted in Table 1 in the presence and absence of a test compound, andselecting a compound that increases the level of expression and/oractivity of the one or more polypeptides relative to the level in theabsence of the compound, as a compound useful for inhibition of tumorgrowth or metastasis.

In each aspect above, the assay may be a cell-based or cell-free assay.In one embodiment, the cell may be a primary cell, e.g., an endothelialcell or a tumor cell, such as a breast tumor cell. In anotherembodiment, the cell is from a cell line, e.g., an endothelial cell lineor a tumor cell line. Endothelial cells and cell lines include, withoutlimitation, HUVEC, HCEC, HGEC, HUV-ST, ECY304, ECV304, and EA.hy926. Thecell may be contacted with the compound in vitro (e.g., in a culturedish) or in an animal (e.g., a transgenic animal or an animal model). Inone embodiment, the detected increase or decrease in expression and/oractivity is statistically significant, e.g., at least p<0.05, e.g.,p<0.01, 0.005, or 0.001. In another embodiment, the detected increase ordecrease is at least about 10%, 20%, 30%, 40%, 50%, 60&, 70%, 80%, 90%,100% or more.

Any desired end-point can be detected in a screening assay, e.g.,binding to the polypeptide, gene or RNA, modulation of the activity ofthe polypeptide, modulation of angiogenesis-related pathways, and/orinterference with binding by a known regulator of a polynucleotide orpolypeptide. Methods of detecting the foregoing activities are known inthe art and include the methods disclosed herein.

Any compound of interest can be screened according to the presentinvention. Suitable test compounds include organic and inorganicmolecules. Suitable organic molecules can include but are not limited tosmall molecules (compounds less than about 1000 Daltons), polypeptides(including enzymes, antibodies, and Fab′ fragments), carbohydrates,lipids, coenzymes, and nucleic acid molecules (including DNA, RNA, andchimerics and analogs thereof) and nucleotides and nucleotide analogs.In particular embodiments, the compound is an antisense nucleic acid, ansiRNA, or a ribozyme that inhibits production of a polypeptide listed inTable 1.

Further, the methods of the invention can be practiced to screen acompound library, e.g., a small molecule library, a combinatorialchemical compound library, a polypeptide library, a cDNA library, alibrary of antisense nucleic acids, and the like, or an arrayedcollection of compounds such as polypeptide and nucleic acid arrays.

In one representative embodiment, the invention provides methods ofscreening test compounds to identify a test compound that binds to apolypeptide listed in Table 1 or functional fragment thereof. Compoundsthat are identified as binding to the polypeptide or functional fragmentcan be subject to further screening (e.g., for modulation ofangiogenesis) using the methods described herein or other suitabletechniques.

Also provided are methods of screening compounds to identify those thatmodulate the activity of a polypeptide listed in Table 1 or functionalfragment thereof. The term “modulate” is intended to refer to compoundsthat enhance (e.g., increase) or inhibit (e.g., reduce) the activity ofthe polypeptide (or functional fragment). For example, the interactionof the polypeptide or functional fragment with a binding partner can beevaluated. As another alternative, physical methods, such as NMR, can beused to assess biological function. Activity of the polypeptides listedin Table 1 or functional fragment can be evaluated by any method knownin the art, including the methods disclosed herein.

Compounds that are identified as modulators of activity can optionallybe further screened using the methods described herein (e.g., forbinding to the polypeptide listed in Table 1 or functional fragmentthereof, polynucleotide or RNA, modulation of mineralization, and thelike). The compound can directly interact with the polypeptide orfunctional fragment, polynucleotide or mRNA and thereby modulate itsactivity. Alternatively, the compound can interact with any otherpolypeptide, nucleic acid or other molecule as long as the interactionresults in a modulation of the activity of the polypeptide or functionalfragment.

As another aspect, the invention provides a method of identifyingcompounds that modulate angiogenesis. In one representative embodiment,the method comprises contacting a polypeptide listed in Table 1 orfunctional fragment thereof with a test compound; and detecting whetherthe test compound binds to the polypeptide or functional fragment and/ormodulates the activity of the polypeptide (or fragment). In anotherexemplary embodiment, the method comprises introducing a test compoundinto a cell that comprises the polypeptide listed in Table 1 orfunctional fragment; and detecting whether the compound binds to thepolypeptide or functional fragment and/or modulates the activity of thepolypeptide or functional fragment in the cell. The polypeptide can beendogenously produced in the cell. Alternatively or additionally, thecell can be modified to comprise an isolated polynucleotide encoding,and optionally overexpressing, the polypeptide or functional fragmentthereof.

The screening assay can be a cell-based or cell-free assay. Further, thepolypeptide listed in Table 1 (or functional fragment thereof) orpolynucleotide can be free in solution, affixed to a solid support,expressed on a cell surface, or located within a cell.

With respect to cell-free binding assays, test compounds can besynthesized or otherwise affixed to a solid substrate, such as plasticpins, glass slides, plastic wells, and the like. For example, the testcompounds can be immobilized utilizing conjugation of biotin andstreptavidin by techniques well known in the art. The test compounds arecontacted with the polypeptide or functional fragment thereof andwashed. Bound polypeptide can be detected using standard techniques inthe art (e.g., by radioactive or fluorescence labeling of thepolypeptide or functional fragment, by ELISA methods, and the like).

Alternatively, the target can be immobilized to a solid substrate andthe test compounds contacted with the bound polypeptide or functionalfragment thereof. Identifying those test compounds that bind to and/ormodulate the polypeptide listed in Table 1 or functional fragment can becarried out with routine techniques. For example, the test compounds canbe immobilized utilizing conjugation of biotin and streptavidin bytechniques well known in the art. As another illustrative example,antibodies reactive with the polypeptide or functional fragment can bebound to the wells of the plate, and the polypeptide trapped in thewells by antibody conjugation. Preparations of test compounds can beincubated in the polypeptide (or functional fragment)-presenting wellsand the amount of complex trapped in the well can be quantitated.

In another representative embodiment, a fusion protein can be providedwhich comprises a domain that facilitates binding of the polypeptide toa matrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with cell lysates (e.g., ³⁵S-labeled) and the test compound,and the mixture incubated under conditions conducive to complexformation (e.g., at physiological conditions for salt and pH). Followingincubation, the beads are washed to remove any unbound label, and thematrix immobilized and radiolabel detected directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of polypeptide listed in Table 1 or functional fragmentthereof found in the bead fraction quantitated from the gel usingstandard electrophoretic techniques.

Another technique for compound screening provides for high throughputscreening of compounds having suitable binding affinity to thepolypeptide of interest, as described in published PCT applicationWO84/03564. In this method, a large number of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with the polypeptidelisted in Table 1 or functional fragment thereof and washed. Boundpolypeptide is then detected by methods well known in the art. Purifiedpolypeptide or a functional fragment can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

With respect to cell-based assays, any suitable cell can be used,including bacteria, yeast, insect cells (e.g., with a baculovirusexpression system), avian cells, mammalian cells, or plant cells. Inexemplary embodiments, the assay is carried out in a cell line thatnaturally expresses the polynucleotide or produces the polypeptide,e.g., endothelial cells or pericytes. Further, in other embodiments, itis desirable to use nontransformed cells (e.g., primary cells) astransformation may alter the function of the polypeptide.

The screening assay can be used to detect compounds that bind to ormodulate the activity of the native polypeptide listed in Table 1 (e.g.,polypeptide that is normally produced by the cell). Alternatively, thecell can be modified to express (e.g., overexpress) a recombinantpolypeptide or functional fragment thereof. According to thisembodiment, the cell can be transiently or stably transformed with apolynucleotide encoding the polypeptide listed in Table 1 or functionalfragment, but is preferably stably transformed, for example, by stableintegration into the genome of the organism or by expression from astably maintained episome (e.g., Epstein Barr Virus derived episomes).In another embodiment, a polynucleotide encoding a reporter molecule canbe linked to a regulatory element of the polynucleotide encoding apolypeptide listed in Table 1 and used to identify compounds thatmodulate expression of the polypeptide.

In a cell-based assay, the compound to be screened can interact directlywith the polypeptide listed in Table 1 or functional fragment thereof(i.e., bind to it) and modulate the activity thereof. Alternatively, thecompound can be one that modulates polypeptide activity (or the activityof a functional fragment) at the nucleic acid level. To illustrate, thecompound can modulate transcription of the gene (or transgene), modulatethe accumulation of mRNA (e.g., by affecting the rate of transcriptionand/or turnover of the mRNA), and/or modulate the rate and/or amount oftranslation of the mRNA transcript.

As a further type of cell-based binding assay, the polypeptide listed inTable 1 or functional fragment thereof can be used as a “bait protein”in a two-hybrid or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al., Cell 72:223 (1993); Madura et al., J. Biol.Chem. 268:12046 (1993); Bartel et al., Biotechniques 14:920 (1993);Iwabuchi et al., Oncogene 8:1693 (1993); and PCT publicationWO94/10300), to identify other polypeptides that bind to or interactwith the polypeptide of the invention or functional fragment thereof.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the polynucleotide that encodes thepolypeptide listed in Table 1 or functional fragment thereof is fused toa nucleic acid encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, optionallyfrom a library of DNA sequences, that encodes an unidentified protein(“prey” or “sample”) is fused to a nucleic acid that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact in vivo, forming a complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter sequence (e.g., LacZ), which is operably linked to atranscriptional regulatory site responsive to the transcription factor.Expression of the reporter can be detected and cell colonies containingthe functional transcription factor can be isolated and used to obtainthe nucleic acid encoding the polypeptide that exhibited binding to thepolypeptide listed in Table 1 or functional fragment.

As another cell-based assay, the invention provides a method ofscreening a compound for modulation of angiogenesis. In particularembodiments, the cell comprises an isolated polynucleotide encoding thepolypeptide listed in Table 1 or functional fragment thereof. Accordingto this embodiment, it is preferred that the isolated polynucleotideencoding the polypeptide or functional fragment is stably incorporatedinto the cell (i.e., by stable integration into the genome of theorganism or by expression from a stably maintained episome such asEpstein Barr Virus derived episomes).

Screening assays can also be carried out in vivo in animals. Thus, asstill a further aspect, the invention provides a transgenic non-humananimal comprising an isolated polynucleotide encoding a polypeptidelisted in Table 1 or functional fragment thereof, which can be producedaccording to methods well-known in the art. The transgenic non-humananimal can be from any species, including avians and non-human mammals.According to this aspect of the invention, suitable non-human mammalsinclude mice, rats, rabbits, guinea pigs, goats, sheep, pigs, andcattle. Suitable avians include chickens, ducks, geese, quail, turkeys,and pheasants.

The polynucleotide encoding the polypeptide or functional fragment canbe stably incorporated into cells within the transgenic animal(typically, by stable integration into the genome or by stablymaintained episomal constructs). It is not necessary that every cellcontain the transgene, and the animal can be a chimera of modified andunmodified cells, as long as a sufficient number of cells comprise andexpress the polynucleotide encoding the polypeptide or functionalfragment so that the animal is a useful screening tool.

Exemplary methods of using the transgenic non-human animals of theinvention for in vivo screening of compounds that modulate angiogenesis,tumor growth, metastasis, and/or the activity of a polypeptide listed inTable 1 comprise administering a test compound to a transgenic non-humananimal (e.g., a mammal such as a mouse) comprising an isolatedpolynucleotide encoding a polypeptide listed in Table 1 or functionalfragment thereof stably incorporated into the genome and detectingwhether the test compound modulates angiogenesis, tumor growth,metastasis, and/or polypeptide activity (or the activity of a functionalfragment).

It is known in the art how to measure these responses in vivo.Illustrative approaches include observation of changes that can bestudied by gross examination (e.g., formation of tubules and bloodvessels), histopathology, cell markers, and enzymatic activity.

Methods of making transgenic animals are known in the art. DNA or RNAconstructs can be introduced into the germ line of an avian or mammal tomake a transgenic animal. For example, one or several copies of theconstruct can be incorporated into the genome of an embryo by standardtransgenic techniques.

In an exemplary embodiment, a transgenic non-human animal is produced byintroducing a transgene into the germ line of the non-human animal.Transgenes can be introduced into embryonal target cells at variousdevelopmental stages. Different methods are used depending on the stageof development of the embryonal target cell. The specific line(s) of anyanimal used should, if possible, be selected for general good health,good embryo yields, good pronuclear visibility in the embryo, and goodreproductive fitness.

Introduction of the transgene into the embryo can be accomplished by anyof a variety of means known in the art such as microinjection,electroporation, lipofection, or a viral vector. For example, thetransgene can be introduced into a mammal by microinjection of theconstruct into the pronuclei of the fertilized mammalian egg(s) to causeone or more copies of the construct to be retained in the cells of thedeveloping mammal(s). Following introduction of the transgene constructinto the fertilized egg, the egg can be incubated in vitro for varyingamounts of time, or reimplanted into the surrogate host, or both. Onecommon method is to incubate the embryos in vitro for about 1-7 days,depending on the species, and then reimplant them into the surrogatehost.

The progeny of the transgenically manipulated embryos can be tested forthe presence of the construct by Southern blot analysis of a segment oftissue. An embryo having one or more copies of the exogenous clonedconstruct stably integrated into the genome can be used to establish apermanent transgenic animal line.

Transgenically altered animals can be assayed after birth for theincorporation of the construct into the genome of the offspring. Thiscan be done by hybridizing a probe corresponding to the polynucleotidesequence coding for the polypeptide or a segment thereof ontochromosomal material from the progeny. Those progeny found to contain atleast one copy of the construct in their genome are grown to maturity.

Methods of producing transgenic avians are also known in the art, see,e.g., U.S. Pat. No. 5,162,215.

In particular embodiments, to create an animal model in which theactivity or expression of a polypeptide listed in Table 1 is decreased,it is desirable to inactivate, replace or knock-out the endogenous geneencoding the polypeptide by homologous recombination with a transgeneusing embryonic stem cells. In this context, a transgene is meant torefer to heterologous nucleic acid that upon insertion within oradjacent to the gene results in a decrease or inactivation of geneexpression or polypeptide amount or activity.

A knock-out of a gene means an alteration in the sequence of a gene thatresults in a decrease of function of the gene, preferably such that thegene expression or polypeptide amount or activity is undetectable orinsignificant. Knock-outs as used herein also include conditionalknock-outs, where alteration of the gene can occur upon, for example,exposure of the animal to a substance that promotes gene alteration(e.g., tetracycline or ecdysone), introduction of an enzyme thatpromotes recombination at a gene site (e.g., Cre in the Cre-lox system),or other method for directing the gene alteration postnatally. Knock-outanimals may be prepared using methods known to those of skill in theart. See, for example, Hogan, et al. (1986) Manipulating the MouseEmbryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

A knock-out construct is a nucleic acid sequence, such as a DNA or RNAconstruct, which, when introduced into a cell, results in suppression(partial or complete) of expression of a polypeptide encoded byendogenous DNA in the cell. A knock-out construct as used herein mayinclude a construct containing a first fragment from the 5′ end of thegene encoding a polypeptide listed in Table 1, a second fragment fromthe 3′ end of the gene and a DNA fragment encoding a selectable markerpositioned between the first and second fragments. It should beunderstood by the skilled artisan that any suitable 5′ and 3′ fragmentsof a gene may be used as long as the expression of the correspondinggene is partially or completely suppressed by insertion of thetransgene. Suitable selectable markers include, but are not limited to,neomycin, puromycin and hygromycin. In addition, the construct maycontain a marker, such as diphtheria toxin A or thymidine kinase, forincreasing the frequency of obtaining correctly targeted cells. Suitablevectors include, but are not limited to, pBLUESCRIPT, pBR322, and pGEM7.

Alternatively, a knock-out construct may contain RNA molecules such asantisense RNA, siRNA, and the like to decrease the expression of a geneencoding a polypeptide listed in Table 1. Typically, for stableexpression the RNA molecule is placed under the control of a promoter.The promoter may be regulated, if deficiencies in the protein ofinterest may lead to a lethal phenotype, or the promoter may driveconstitutive expression of the RNA molecule such that the gene ofinterest is silenced under all conditions of growth. While homologousrecombination between the knock-out construct and the gene of interestmay not be necessary when using an RNA molecule to decrease geneexpression, it may be advantageous to target the knock-out construct toa particular location in the genome of the host organism so thatunintended phenotypes are not generated by random insertion of theknock-out construct.

The knock-out construct may subsequently be incorporated into a viral ornonviral vector for delivery to the host animal or may be introducedinto embryonic stem (ES) cells. ES cells are typically selected fortheir ability to integrate into and become part of the germ line of adeveloping embryo so as to create germ line transmission of theknock-out construct. Thus, any ES cell line that can do so is suitablefor use herein. Suitable cell lines which may be used include, but arenot limited to, the 129J ES cell line or the Jl ES cell line. The cellsare cultured and prepared for DNA insertion using methods well-known tothe skilled artisan (e.g., see Robertson (1987) In: Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. IRLPress, Washington, D.C.; Bradley et al., Curr. Topics Develop. Biol.20:357 (1986); Hogan et al., (1986) Manipulating the Mouse Embryo: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

Insertion of the knock-out construct into the ES cells may beaccomplished using a variety of methods well-known in the art,including, for example, electroporation, microinjection, and calciumphosphate treatment. For insertion of the DNA or RNA sequence, theknock-out construct nucleic acids are added to the ES cells underappropriate conditions for the insertion method chosen. If the cells areto be electroporated, the ES cells and construct nucleic acids areexposed to an electric pulse using an electroporation machine(electroporator) and following the manufacturer's guidelines for use.After electroporation, the cells are allowed to recover under suitableincubation conditions. The cells are then screened for the presence ofthe knockout construct.

Each knock-out construct to be introduced into the cell is firsttypically linearized if the knock-out construct has been inserted into avector. Linearization is accomplished by digesting the knock-outconstruct with a suitable restriction endonuclease selected to cut onlywithin the vector sequence and not within the knock-out constructsequence.

Screening for cells which contain the knock-out construct (homologousrecombinants) may be done using a variety of methods. For example, asdescribed herein, cells can be processed as needed to render DNA in themavailable for hybridization with a nucleic acid probe designed tohybridize only to cells containing the construct. For example, cellularDNA can be probed with ³²P-labeled DNA which locates outside thetargeting fragment. This technique can be used to identify those cellswith proper integration of the knock-out construct. The DNA can beextracted from the cells using standard methods (e.g., see, Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor,N.Y., 1989)). The DNA may then be analyzed by Southern blot with a probeor probes designed to hybridize in a specific pattern to genomic DNAdigested with one or more particular restriction enzymes.

Once appropriate ES cells are identified, they are introduced into anembryo using standard methods. They can be introduced usingmicroinjection, for example. Embryos at the proper stage of developmentfor integration of the ES cell to occur are obtained, such as byperfusion of the uterus of pregnant females. For example, mouse embryosat 3-4 days development can be obtained and injected with ES cells usinga micropipet. After introduction of the ES cell into the embryo, theembryo is introduced into the uterus of a pseudopregnant female mouse.The stage of the pseudopregnancy is selected to enhance the chance ofsuccessful implantation. In mice, 2-3 days pseudopregnant females areappropriate.

Germline transmission of the knockout construct may be determined usingstandard methods. Offspring resulting from implantation of embryoscontaining the ES cells described above are screened for the presence ofthe desired alteration (e.g., knock-out of the polypeptide listed inTable 1). This may be done, for example, by obtaining DNA from offspring(e.g., tail DNA) to assess for the knock-out construct, using knownmethods (e.g., Southern analysis, dot blot analysis, PCR analysis). See,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2ndEd. (Cold Spring Harbor, N.Y., 1989). Offspring identified as chimerasmay be crossed with one another to produce homozygous knock-out animals.

Mice are often used as animal models because they are easy to house,relatively inexpensive, and easy to breed. However, other knock-outanimals may also be made in accordance with the present invention suchas, but not limited to, monkeys, cattle, sheep, pigs, goats, horses,dogs, cats, guinea pigs, rabbits and rats. Accordingly, appropriatevectors and promoters well-known in the art may be selected and used togenerate a transgenic animal deficient in expression of a polypeptidelisted in Table 1.

In another embodiment, animal models may be created using animals thatare not transgenic. For example, tumor models (e.g., created bydelivering tumorigenic cells into immunocompromised animals) can be usedto study the effects of regulators of angiogenesis on tumor growth andmetastasis. In another example, tumorigenic cells that overexpress orunderexpress a polypeptide listed in Table 1 can be delivered to ananimal under conditions in which tumors develop from the cells. Tumorgrowth in the animals can be compared to tumor growth in animalscontaining cells that do not overexpress or underexpress thepolypeptide.

VIII. Pharmaceutical Compositions

As a further aspect, the invention provides pharmaceutical formulationsand methods of administering the same to achieve any of the therapeuticeffects (e.g., inhibition or stimulation of angiogenesis) discussedabove. The pharmaceutical formulation may comprise any of the reagentsdiscussed above in a pharmaceutically acceptable carrier, e.g., apolynucleotide encoding a polypeptide listed in Table 1 or a fragmentthereof, a polypeptide listed in Table 1 or fragment thereof, anantibody against a polypeptide listed in Table 1, an antisenseoligonucleotide, an siRNA molecule, a ribozyme, an aptamer, apeptidomimetic, a small molecule, or any other compound that modulatesthe activity of a polypeptide listed in Table 1, including compoundsidentified by the screening methods described herein.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, i.e., the material can beadministered to a subject without causing any undesirable biologicaleffects such as toxicity.

The formulations of the invention can optionally comprise medicinalagents, pharmaceutical agents, carriers, adjuvants, dispersing agents,diluents, and the like.

The compounds of the invention can be formulated for administration in apharmaceutical carrier in accordance with known techniques. See, e.g.,Remington, The Science And Practice of Pharmacy (9^(th) Ed. 1995). Inthe manufacture of a pharmaceutical formulation according to theinvention, the compound (including the physiologically acceptable saltsthereof) is typically admixed with, inter alfa, an acceptable carrier.The carrier can be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which can contain from 0.01 or 0.5% to 95% or 99% by weight ofthe compound. One or more compounds can be incorporated in theformulations of the invention, which can be prepared by any of thewell-known techniques of pharmacy.

A further aspect of the invention is a method of treating subjects invivo, comprising administering to a subject a pharmaceutical compositioncomprising a compound of the invention in a pharmaceutically acceptablecarrier, wherein the pharmaceutical composition is administered in atherapeutically effective amount. Administration of the compounds of thepresent invention to a human subject or an animal in need thereof can beby any means known in the art for administering compounds.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular including skeletal muscle, cardiac muscle,diaphragm muscle and smooth muscle, intradermal, intravenous,intraperitoneal), topical (i.e., both skin and mucosal surfaces,including airway surfaces), intranasal, transdermal, intraarticular,intrathecal, and inhalation administration, administration to the liverby intraportal delivery, as well as direct organ injection (e.g., intothe liver, into the brain for delivery to the central nervous system,into the pancreas, or into a tumor or the tissue surrounding a tumor).The most suitable route in any given case will depend on the nature andseverity of the condition being treated and on the nature of theparticular compound which is being used.

For injection, the carrier will typically be a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.). Forother methods of administration, the carrier can be either solid orliquid.

For oral administration, the compound can be administered in soliddosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Compounds can beencapsulated in gelatin capsules together with inactive ingredients andpowdered carriers, such as glucose, lactose, sucrose, mannitol, starch,cellulose or cellulose derivatives, magnesium stearate, stearic acid,sodium saccharin, talcum, magnesium carbonate and the like. Examples ofadditional inactive ingredients that can be added to provide desirablecolor, taste, stability, buffering capacity, dispersion or other knowndesirable features are red iron oxide, silica gel, sodium laurylsulfate, titanium dioxide, edible white ink and the like. Similardiluents can be used to make compressed tablets. Both tablets andcapsules can be manufactured as sustained release products to providefor continuous release of medication over a period of hours. Compressedtablets can be sugar coated or film coated to mask any unpleasant tasteand protect the tablet from the atmosphere, or enteric-coated forselective disintegration in the gastrointestinal tract. Liquid dosageforms for oral administration can contain coloring and flavoring toincrease patient acceptance.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the compound in a flavored base, usually sucrose andacacia or tragacanth; and pastilles comprising the compound in an inertbase such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the compound, which preparations are preferably isotonicwith the blood of the intended recipient. These preparations can containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient. Aqueousand non-aqueous sterile suspensions can include suspending agents andthickening agents. The formulations can be presented in unit\dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, saline orwater-for-injection immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound of theinvention, in a unit dosage form in a sealed container. The compound orsalt is provided in the form of a lyophilizate which is capable of beingreconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection thereof into a subject.The unit dosage form typically comprises from about 10 mg to about 10grams of the compound or salt. When the compound or salt issubstantially water-insoluble, a sufficient amount of emulsifying agentwhich is pharmaceutically acceptable can be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier. Onesuch useful emulsifying agent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These can be prepared by admixing thecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the farm of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which can be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration can be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration can also be delivered byiontophoresis (see, for example, Tyle, Pharm. Res. 3:318 (1986)) andtypically take the form of an optionally buffered aqueous solution ofthe compound. Suitable formulations comprise citrate or bis\tris buffer(pH 6) or ethanol/water and contain from 0.1 to 0.2M of the compound.

The compound can alternatively be formulated for nasal administration orotherwise administered to the lungs of a subject by any suitable means,e.g., administered by an aerosol suspension of respirable particlescomprising the compound, which the subject inhales. The respirableparticles can be liquid or solid. The term “aerosol” includes anygas-borne suspended phase, which is capable of being inhaled into thebronchioles or nasal passages. Specifically, aerosol includes agas-borne suspension of droplets, as can be produced in a metered doseinhaler or nebulizer, or in a mist sprayer. Aerosol also includes a drypowder composition suspended in air or other carrier gas, which can bedelivered by insufflation from an inhaler device, for example. SeeGanderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood(1987); Gonda (1990) Critical Reviews in Therapeutic Drug CarrierSystems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth.27:143 (1992). Aerosols of liquid particles comprising the compound canbe produced by any suitable means, such as with a pressure-drivenaerosol nebulizer or an ultrasonic nebulizer, as is known to those ofskill in the art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solidparticles comprising the compound can likewise be produced with anysolid particulate medicament aerosol generator, by techniques known inthe pharmaceutical art.

Alternatively, one can administer the compound in a local rather thansystemic manner, for example, in a depot or sustained-releaseformulation.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same can be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed can be of anyconventional composition and can either contain cholesterol or can becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced can be reduced insize, as through the use of standard sonication and homogenizationtechniques.

The liposomal formulations containing the compounds disclosed herein orsalts thereof, can be lyophilized to produce a lyophilizate which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

In the case of water-insoluble compounds, a pharmaceutical compositioncan be prepared containing the water-insoluble compound, such as forexample, in an aqueous base emulsion. In such an instance, thecomposition will contain a sufficient amount of pharmaceuticallyacceptable emulsifying agent to emulsify the desired amount of thecompound. Particularly useful emulsifying agents include phosphatidylcholines and lecithin.

In particular embodiments, the compound is administered to the subjectin a therapeutically effective amount, as that term is defined above.Dosages of pharmaceutically active compounds can be determined bymethods known in the art, see, e.g., Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.). The therapeutically effectivedosage of any specific compound will vary somewhat from compound tocompound, and patient to patient, and will depend upon the condition ofthe patient and the route of delivery. As a general proposition, adosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy,with all weights being calculated based upon the weight of the compound,including the cases where a salt is employed. Toxicity concerns at thehigher level can restrict intravenous dosages to a lower level such asup to about 10 mg/kg, with all weights being calculated based upon theweight of the compound, including the cases where a salt is employed. Adosage from about 10 mg/kg to about 50 mg/kg can be employed for oraladministration. Typically, a dosage from about 0.5 mg/kg to 5 mg/kg canbe employed for intramuscular injection. Particular dosages are about 1μmol/kg to 50 μmol/kg, and more particularly to about 22 μmol/kg and to33 μmol/kg of the compound for intravenous or oral administration,respectively.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) can be employed over avariety of time intervals (e.g., hourly, daily, weekly, monthly, etc.)to achieve therapeutic effects.

The present invention finds use in veterinary and medical applications.Suitable subjects include both avians and mammals, with mammals beingpreferred. The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys, and pheasants. The term“mammal” as used herein includes, but is not limited to, humans,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.Human subjects include neonates, infants, juveniles, and adults. Inother embodiments, the subject is an animal model of bone disease.

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

Example 1 Experimental Methods

Breast Tissue Source:

The frozen tissues and tumors used were obtained from the LinebergerComprehensive Cancer Center Tissue Procurement and Analysis Core andhave been procured from patients who were appropriately informed and whohave consented to having their tissue procured for research. The tissuewas obtained from primary breast tumors in patients who were not treatedwith neoadjuvant chemotherapy, or from patients without cancerundergoing reduction mammoplasty. The breast tumors used formicrodissection were ER⁺, Her2/neu⁻ (luminal A immunophenotype).

Immunohistochemistry for Laser Capture Microdissection:

Portions of snap frozen breast tissue were fixed in OCT compound andsectioned at −35° C. on a cryostat at 8 μm onto polyethylene naphthalatemembrane glass slides (Arcturus Bioscience, Mt View, Calif., catalogue#LCM0522). RNAse free technique was used throughout the procedure andbuffers and alcohol solutions were used fresh each time. Slides werefixed in acetone for 2 minutes at 4° C. and rinsed in Hank's balancedsalt solution (HBSS) (Gibco, Grand Island, N.Y.). The slides wereincubated with a mouse-antihuman antibody to factor VIII-related antigen(BioGenex, San Ramon, Calif., catalogue # MU016-UC) at a 1:6 dilutionfor 7 minutes at 4° C. The IHC was performed with the DakoCytomationLSAB 2 system (Carpinteria, Calif.), a three step streptavidin-biotinsystem with the following modifications. After washing in HBSS, thebiotinylated link was incubated for 5 minutes at room temperature.BCIP/NBT alkaline phosphatase developer (Vector Labs, Burlingame,Calif.) was used at a very high concentration (3 drops/300 μl buffer)and incubated for 10-15 minutes at 4° C. Slides were dehydrated in 75%ETOH for 30 seconds, 95% ETOH for 30 seconds, and 100% ETOH for 2minutes (Arcturus, Mountain View, Calif.). Protector RNAse Inhibitor(Roche, Indianapolis, Ind.) was added at a 1:10 dilution to all buffersused in the staining process. The slides were placed on dry ice untilmicrodissection, which occurred the same day as the immunohistochemistry(MC).

Laser Capture Microdissection:

Microdissection immediately followed tissue preparation. Laser capturemicrodissection was performed on a Leica Laser Microdissection System.Tissues to be microdissected were viewed through a video microscope andthe position of the slide was adjusted so that the desired cells wereunder the targeting light. Activation of the UV laser cut the tissuearound the groups of cells of interest. The cut tissue was thentransported by gravity to an eppendorf tube that contained 25 μl of RNAextraction buffer from the Picopure RNA Extraction Kit™ (Arcturus,Mountain View, Calif.). In order to maintain RNA integrity, slides werekept on dry ice until microdissection, and microdissection was performedfor no longer than 15 minutes per slide. Fifteen slides weremicrodissected per sample. RNA was then extracted with the ArcturusPicopure RNA Extraction Kit™ (Arcturus, Mountain View, Calif.) asdescribed in the manufacturer's instructions and DNAse I treated.

Amplification of RNA:

RNA amplification was performed using a two round amplification system.The first round employed the RiboAmp® HS RNA Amplification Kit(Arcturus, Mountain View, Calif.). Five hundred ng from the first roundof amplification was then put into the Agilent Low-Input FluorescentLinear RNA Amplification Kit™ (Palo Alto, Calif.). This second roundemployed a T7 polymerase amplification that incorporated the fluorescentprobe in preparation for microarray analyses.

Analyses of RNA Integrity:

RNA integrity was checked after the first round of amplification priorto each microarray experiment using RT-PCR detection of genes ofdifferent abundance levels and demonstration of intact, full-length cDNApreparations with the cDNA Integrity Kit (KPL, Gaithersburg, Md.). Thelatter system utilizes primer sets and target genes that allowevaluation of in-process or double-stranded cDNA for the presence offull-length and extended cDNA transcripts. Primer sets amplify regionsof the 3′ and 5′ ends of the housekeeping genes GAPDH and the lowexpressed ADP ribosylation factor I gene. Generation of product usingthe 3′ primer sets indicate that the gene is expressed in the system,and amplimer production using the 5′ primer sets indicate full length,intact cDNA.

Measurement of Amplification Bias:

MDA-MB-435 breast cancer cells were plated (2.5×10⁶ cells) in 75 cm²flasks or 100 mm plates in DMEM with 10% fetal bovine serum and 100 U ofpenicillin-streptomycin (Gibco). After 48 hours, total RNA was extractedusing the Qiagen RNEASY Kit and purified with QIAquick PCR PurificationKit (Qiagen, Valencia, Calif.). Samples underwent only one round ofamplification (Group A) or two rounds of amplification (Group B).Correlation coefficients among arrays were compared with interclasscorrelation (Hu et al., Biatechniques 38:121 (2005)).

Microarray Experiments:

Synthesis of labeled cDNA was performed as described previously withreference cDNA that is the Stratagene Human Universal Reference (Hu etal., Biotechniques 38:121 (2005)) labeled with Cy3-dUTP and sample cDNAslabeled with Cy5-dUTP. Microarray hybridizations were performed usingAgilent Human oligonucleotide (Custom designed Agilent 1Av1-based forcell lines and Agilent 44k for vessel-dissected specimens) microarraysas previously described (Hu et al., Biotechniques 38:121 (2005)).Technical replicates (which refer to using the same RNA from one tumoron two microarrays) were performed for all vessel-dissected specimens.

Data Normalization, Preprocessing, and Statistics:

Gene expression values were quantified using the log₂ ratio of theLowess normalized red channel intensity versus green channel intensity(Yang et al., Nucleic Acids Res. 30:e15 (2002)). The UNC Microarraydatabase (genome.unc.edu) was used to perform the filtering andpreprocessing, and all data are available from the UMD and have beendeposited into the GEO under the accession number of GSE7413. Atwo-class SAM (Significance Analysis of Microarrays,www-stat.stanford.edu/˜tibs/SAM) (Storey, J. R. Stat. Soc. Series B:479(2002); Tusher et al., Proc. Natl. Acad. Sci. USA 98:5116 (2001)) wasperformed to identify significantly differentially expressed genesbetween all 5 tumor vascular samples versus all 5 normal vascularsamples. Each sample had a technical replicate array, thus there were 10arrays in each group that were used for the SAM. In order to identifydifferentially expressed genes that encode potential membrane orsecreted proteins, Gene Cards (www.genecards.org/index.shtml) wassearched to identify the potential subcellular location for geneswith >4 fold increased expression.

In order to interpret the gene lists derived from the results of SAM,and convert the gene list into biological themes, EASE (the ExpressionAnalysis Systematic Explorer, david.abcc.ncifcrf.gov) analysis wasapplied.

Identity of Cell Types in Microdissected Vessel Cells:

The cell types comprising the microdissected vessels were identified byanalyzing gene expression for genes known to be selectively expressed inspecific populations of cells (endothelial, hematopoietic, pericytes,and epithelial) and comparing gene expression profiles from the vascularcell specimens to endothelial cell cultures in vitro and breasttumor-derived cells cultures in vitro. Human endothelial cell total RNAswere purchased from Cell Application Incorporation (San Diego, Calif.).Total RNA was purified from breast cancer cell lines using the QiagenRNAeasy Kit. RNA integrity was determined using the RNA 6000 NanoLabChip Kit and Agilent 2100 Bioanalyzer. Genes specific forendothelium, previously characterized TEMs, hematopoietic markers,pericyte markers, and luminal epithelium were analyzed and the datadisplayed using Java Treeview (Saldanha, Bioinformatics 20:3246 (2004)).

Confirmation of Vascular Origin of Vascular Marker Genes:

To validate the vascular origin of the genes associated with tumorendothelium obtained by immuno-LCM, immunohistochemistry was performedwith antibodies to select gene transcripts and compared with staining onsubsequent sections stained with antibodies to factor VIII-relatedantigen on paraffin embedded ER⁺, Her2/neu⁻ breast tumors.

Commercially Available Antibodies:

Rabbit polyclonal antibody to SFRP-2 (H-140) (Santa Cruz Biotechnology,Santa Cruz, Calif., catalogue # sc-13940) was used at 1:150 dilution;Rabbit polyclonal to FAP/fibroblast activation protein, alpha-Stalkregion (Abeam, Cambridge, Mass., catalogue # Ab28244) was used at 1:600dilution; Mouse monoclonal antibody to JAK3 (Genetex Inc., San Antonio,Tex., catalogue # GTX23301) was used at 1:100 dilution; Mouseanti-Hep27(17) (DHRS2) antibody, a gift from Dr. Franco Gabrielli(Università di Pisa, Pisa, Italy), was used at 1:1000 dilution;Mouse-antihuman antibody to factor VIII-related antigen (BioGenex, SanRamon, Calif., catalogue # MU016-UC), was used at 1:100 dilution; Mousemonoclonal anti-human CD-19 (AbD Serotec, Raleigh, N.C., catalogue #MCA2454T) was used at 1:200).

Antibody Generation Methods:

Peptides to the SLTRK6 (Cys-SRPRKVLVEQTKNEYFELKANLHAEPDYLEVLEQQT (SEQ IDNO:8)) and SMPD3 (TSKSSGQKGRKELLKGNGRRIDYMLHC (SEQ ID NO:9)) proteinswere synthesized and conjugated to keyhole limpet hemocyanin (KLH) forthe immunizations of rabbits. New Zealand White Rabbits (5-6 lbs) wereimmunized three times with 200 μg of the peptide conjugate mixed withFreund's Complete Adjuvant for the primary immunization. Freund'sIncomplete Adjuvant was used for all booster immunizations. The route ofinjection was subcutaneous and intramuscular at multiple sites. Sera wascollected from blood sampling after the third immunization. SLITRK6antibody was used at 1:5000 dilution and SMPD3 antibody was used at1:1000 dilution.

Immunohistochemistry on Paraffin-Embedded Breast Tumor and NormalSamples:

The tissue was sectioned at 8 μm onto Superfrost plus slides. Slideswere dewaxed by immersing in xylene for 5 minutes twice. Slides werehydrated in 100% ETOH, 95% ETOH for 3 minutes each. Slides were quenchedin 3% H₂O₂ (DakoCytomation, LSAB2 HRP Kit, Carpinteria, Calif.) for 10minutes, rinsed in 70% ETOH for 3 minutes, and then PBS for 3 minutes.Citra buffer (BioGenex, San Ramon, Calif.) was warmed in a 60° C. ovenand slides were immersed in citra buffer at 100° C. in a rice steamerfor 30 minutes. Slides were rinsed in PBS for 3 minutes and then markedwith a PAP pen. 100 μl-200 μl of primary antibody was applied and slideswere placed in a sealed box in a 4° C. cold room overnight. Slides werethen rinsed in PBS for 3 minutes, and 1-2 drops of biotinylatedsecondary antibody (DakoCytomation, LSAB2 HRP Kit) was added to eachslide for 20 minutes. Slides were rinsed in PBS for 3 minutes and 1-2drops of streptavidin-HRP (DakoCytomation LSAB2 HRP Kit) was applied for20 minutes. 1-2 drops of DAB complex was applied and slides were placedin a dark drawer for approximately 10 minutes. Slides were rinsed indistilled water for 3 minutes and counterstained with trypan blue(Sigma, St Louis, Mo.) for 30-45 seconds. Slides were rinsed in PBS,dehydrated through graded alcohol and xylene, and Cytoseal XYL(Richard-Allan, Kalamazoo, Mich.) and cover slides were applied. Anegative control without primary antibody was performed for allexperiments, and the positive control was factor VIII-related antigen.

Evaluation of Differential Protein Expression of Vascular Genes BetweenBreast Tumor and Normal Breast Tissue:

Once the vascular genes were confirmed to localize to endothelium, itwas next evaluated whether differential mRNA expression correlated withdifferential protein expression using immunohistochemistry on paraffinembedded breast tumors and normal breast tissue.

Selection of Breast Tumors:

Three groups of formalin-fixed paraffin embedded breast tumors were usedand designated as luminal A, basal, or Her2/neu based on theirimmunophenotypes (Livasy et al., Mod. Pathol. 19:264 (2005)) (“luminalA” ER positive, Her2/neu negative; “basal” ER negative, PR negative,HER2/neu negative; ck5/6 positive or EGFR positive; and “Her2/neu” ERnegative, PR negative, Her2/neu positive) as well as normal breasttissue from reduction mammoplasty. Normal breast tissues were firststained with antibody to factor VIII-related antigen, and only tissuethat had vessels in the sample were used. ER negative, PR negative,Her2/neu negative tumors were stained for CK5/6 antibody (clone 05/16B41:10 dilution, Boehringer Mannheim, Indianapolis) as previouslydescribed (Livasy et al., Mod. Pathol. 19:264 (2005)) and EGFR antibody(clone pharmDx, DakoCytomation Carpinteria, Calif.) per manufacturer'sinstructions to further define the basal phenotype.

Immunohistochemistry Scoring:

A single board-certified pathologist (CAL) scored each tissue sectionfor FAP, SFRP2, JAK3, SMPD3, SLITRK6, DHRS2 and CD19 expression based ona scoring system that measured intensity of stain in endothelium as:(Vessel Intensity Score) 0, none; 1, borderline; 2, weak; 3,moderate/strong, and percent positive endothelial cells staining as: 0,none; 1, 1-24%; 2, 25-49%; 3, 50-74%; 4, 75-100%. Differences in theVessel Intensity Score between tumors and normal tissue were thendichotomized and evaluated, where a “high” score was 3 and a low scorewas 0-2. To further define angiogenesis expression, expression wasdichotomized as high (3+ intensity and 0.75% positive cells) and nothigh (0, 1, or 2 intensity and/or <75% positive cells), and this wasdesignated as the Angiogenesis Score. Fisher's exact test was used totest for possible differences in proportions (or percentages) ofexpression, categorized as either ‘high’ or ‘low’ for both AngiogenesisScore and Vessel Intensity Score between luminal A vs. normal, Her2neuvs. normal, and basal vs. normal tissue. Statistical analyses wereperformed using SAS statistical software, Versions 9.1, SAS InstituteInc., Cary, N.C.

Example 2 Identification of Genes Differentially Expressed in BreastTumor Vessels

Vessel isolation and microarray analysis: In order to study differencesin gene expression between tumor and normal vessels, rapid IHC wasperformed with antibodies to factor VIII-related antigen, followed bylaser capture microdissection (LCM) of vascular cells from 5 luminal Abreast tumors and 5 normal breast tissue specimens from reductionmammoplasty. Immunostaining according to the rapid IHC protocol requiresonly 30-35 minutes from fixation to LCM. The quality of staining wasexcellent, the vascular cells were easily identified, and LCM wasperformed successfully (FIG. 1).

RNA amplification was performed using a two round amplification system.RNA integrity was evaluated after the first round of amplification. Theextracted RNA maintained its integrity as shown by RT-PCR detection ofgenes of different abundance levels (FIG. 2). No signals were observedafter amplification of the negative control (RNA extraction bufferwithout the microdissected sample, data not shown). RNA integrity waschecked on all samples prior to microarray hybridization and onlysamples that maintained RNA integrity were used for microarray analyses.

To estimate the amplification bias, one round of amplified RNA wascompared to two rounds of amplification of RNA extracted from humanMDA-MB-435 breast cancer cells grown in vitro. When both amplified andunamplified RNA were hybridized to 44,000 element Agilent longoligonucleotide DNA microarrays, correlation coefficients ranged from0.95-0.97 among technical replicates.

Confirmation of Vascular Cell Identity and Purity:

Genes specific to endothelium were uniformly and highly expressed in thevascular cell specimens and endothelial cell lines with significantlylower expression seen in the breast tumor cell lines, confirming thatthe vascular cell samples were highly enriched for endothelium (FIG. 3).

Tumor endothelial markers 1, 2, 4, 5, 6, 7, 7R, 8 (previously reportedto be differentially expressed between colon tumor and normalendothelium) (St. Croix, Science 289:1197 (2000)) were highly expressedin both the tumor and normal vascular cells when compared relative tothe low expression seen in the breast tumor cell lines (FIG. 3).Previously reported breast specific tumor vascular genes HEY1, Col4A2,C4A, SPARCL1, SNAIL1 (Parker et al., Cancer Res. 64:7857 (2004)) werealso similarly highly expressed in the samples of both tumor and normalvascular cells, with low expression in the breast tumor cell lines (FIG.3). These results suggest that these are markers of breast endothelium,but their expression was not consistently higher in tumor vs. normalvascular cells.

Platelet derived growth factor receptor beta (PDGFR-β), a pericytemarker, was highly expressed in the vascular cells samples, whichconfirmed the presence of pericytes (FIG. 3). There was high expressionof genes specific to luminal breast tumor epithelium in the breastcancer cell lines, with low expression in the vascular cell samples andendothelial cell lines (FIG. 3). This confirmed enrichment forendothelial cells and pericytes without high levels of expression ofepithelial-associated genes.

Expression of hematopoietic markers in the vascular cells samples wassimilar to the expression in endothelial cell lines in vitro (FIG. 3).CD45 (leukocytes) and CD22 (B cells) had low expression in LCM vesselsand endothelial cell lines. CD14 (macrophages) and CD5 (T cells) wereincreased in both the vascular cell samples and the endothelial celllines. This could be explained by the presence of RNA from macrophagesand T cells in the vascular cell samples. Alternatively, it is possiblethat CD14 and CD5 were expressed on endothelial cells, as there isprevious evidence for monocyte origin of vascular cell precursors(Coukos et al., Br. J. Cancer 92:1182 (2005)) and expression of CD14 inendothelial cells (Jersmann, Immunol. Cell Biol. 83:462 (2005)); CD14was also elevated in a previous report of microdissected ovarian tumorendothelium (Buckanovich et al., Cancer Biol. Ther. 5:635 (2006)), andCD5 has also previously been reported to be present on vascularendothelium (Gogolin-Ewens et al., Eur. J. Immunol. 19:935 (1989)).

Supervised Analysis of Tumor Versus Normal Vessels:

Using Significance Analysis of Microarray (SAM), differentiallyexpressed genes between tumor and normal vascular cells were identified.1176 genes differentially expressed were found with a median number offalse significant=7.76, of which 368 were increased. In order tointerpret the gene list derived from SAM and convert the gene list intobiological themes, the Expression Analysis Systematic Explorer (EASE)was applied. When examining Bonferonni adjusted results, it was foundthat the extracellular matrix ontology category was increased in tumorvascular cells, while the ribosome ontology category was decreased,demonstrating a separate biological response.

Confirmation of Vascular Origin of Vascular Marker Genes:

To validate the vascular origin of the genes associated with tumorendothelium obtained by immuno-LCM, IHC was performed on paraffinembedded luminal A human breast tumors. Since the goal was to identifyhighly differentially expressed genes, the first focus was on the 55genes that had >4 fold increased expression in tumor vessel cells (Table1). From this list, Gene Cards (www.genecards.org/index.shtml) wassearched to identify the potential subcellular location for geneswith >4 fold increased expression, and focused on some of the genes thatpotentially encode membrane proteins (FAP, JAK3, SMPD3, SLITRK6, CD19),and a secreted protein (SFRP2). These would offer particularly good drugtargets due to their accessibility. Also chosen was a gene that hasrecently been described to be expressed in endothelium in vitro (DHRS2)(Shafqat et al., Cell. Mol. Life Sci. 63:1205 (2006)).

Antibodies to factor VIII-related antigen were used for a positivecontrol to identify endothelium, and on subsequent sections, IHC wasperformed with antibodies to FAP (fibroblast activation protein, alpha),SFRP2 (Secreted frizzled-related protein 2), JAK3 (Janus kinase 3),SMPD3 (neutral sphingomyelinase 2), SLITRK6, DHRS2(Dehydrogenase/reductase (SDR family) member 2, also known as Hep27),and CD19.

Antibodies to FAP, SFRP2, JAK3, SMPD3, SLITRK6, and DHRS2 all showedstaining with cellular localization in endothelium (FIG. 4), as well astumor stroma and tumor epithelium. CD19, a B-cell marker, did notlocalize to endothelium. Therefore 6/7 vascular marker genes identifiedby immuno-LCM that were studied appear to be validated of vascularorigin.

Example 3 Evaluation of Differential Protein Expression of VascularGenes Between Breast Tumor Vessels and Normal Breast Vessels

For the six genes validated to be of vascular origin, it was nextevaluated whether differential mRNA expression correlated withdifferential protein expression using IHC on paraffin embedded normal,luminal A, Her2/neu, and basal tumors. Significant differential proteinexpression for SLTRK6 and DHRS2 was not detected, possibly because therewas very high staining in both the tumor endothelium and normalendothelium (data not shown). For SMPD3 there was no difference in theAngiogenesis Score for luminal A versus normal, but there was anincrease in the Vessel Intensity Score comparing luminal A versus normal(15/16 (94%) vs. 6/10 (60%) P=0.05). JAK3 had higher staining in luminalA and Her2/neu tumors compared to normal (p=0.01 and p=0.006respectively, FIG. 5D) and was nearly statistically significant for theAngiogenesis score (p=0.11, FIG. 5C). Basal tumors had very lowexpression of JAK3 (FIG. 5C, 5D). For FAP, the Angiogenesis Scores weresignificantly higher in the luminal A, Her2/neu and basal tumorscompared to normal (p=0.04, p=0.03, and p=0.03 respectively, see FIG.5A). For SFRP2, the Angiogenesis Score was significantly higher inluminal A tumors and basal tumors compared to normal, (p=0.03 and p=0.02respectively) with near significance in Her2/neu tumors (***p=0.10).This appears to validate the original discovery of differential geneexpression in luminal A versus normal vessel cells on a second sampleusing a different platform (IHC).

Example 4 Angiogenic Function of SFRP2

Chick Chorioallantoic Membrane (CAM) Assay:

To determine whether SFRP2 induces angiogenesis in vivo, fertilizedchicken eggs (NC State University Chicken Research Farm) were incubatedat 100° F. on an egg turner for 4 days. On day 4, the eggs were crackedinto sterile Petri dishes and incubated at 99° F. in 3% CO₂, 65%humidity. For application of drug onto the CAM, Whatman grade 1 filterpaper was cut into circles with a 6 mm diameter paper punch andautoclaved. To decrease inflammatory effects of the disk, the discs weresoaked in 1 ml of 3.0 mg/ml cortisone acetate in absolute ETOH and airdried for 60 min in a laminar flow hood. On day 8, 5 disks per egg wereplaced on the outer third of the CAM, 2-3 mm from a vessel. Control PBS7 μl was added to the discs for the control CAMs, and SFRP2 100 ng/7 μlPBS was added to the disks for the treated CAMs (n=5 control disks and 5SFRP2-treated disks). The CAMs were evaluated under a stereomicroscopeon day 3 after disk placement. Pictures were taken with a Wild M-4 70Macrosystem, and angiogenesis was quantified using Metamorph Softwarewith an angiogenesis module. To investigate whether SFRP2 inducesangiogenesis in vivo, SFRP2 impregnated pellets were implanted on thedeveloping CAM on day 8. After 3 days, SFRP2 induced angiogenesis on theCAM with a statistically significant increase in number of branch points(0.010), segments (0.013), tube percent area covered (0.004), total tubearea (0.008), and total tube length (0.008) (FIG. 6).

Scratch Wound Assay:

The migration properties of SFRP2 on mouse endothelial cells (MEC) cellswere evaluated using a scratch wound assay. Mouse endothelial cells wereplated at 10,000 cells/well into a 96 well plate and allowed to becomeconfluent in DMEM with 10% FBS. The cells were quiesced in DMEM withoutserum for 18 hours. The wound was formed using a 1 ml pipette tip and a0.7 pM-700 pM dose curve of mouse recombinant SFRP2 (US Biologicals,Swampscott) was added to the cells. Each concentration was performed intriplicate and the experiment was repeated three times with similarresults. Migration was measured from 16 to 32 hours. Migration distancewas measured at each time point. Statistical differences between SFRP2and control were evaluated with an unpaired two-tailed Student's t-test,with p<0.05 being significant. SFRP2 increased endothelial cellmigration in the picomolar concentration (p<0.01 at 16 hours, p<0.001 at19 hours) (FIG. 7).

Tube Formation Assay:

The tube formation properties of SFRP2 on mouse endothelial cells (MEC)cells were evaluated using an endothelial cell tube formation assay.ECMATRIX (Chemicon) was thawed, diluted and solidified in a 96 wellplate according to the manufacturer's instructions. 1×10⁴ cells/well in150 μl of DMEM (celigro) with 10% FBS (HyClone) and a concentrationrange (7-7000 pM) of SFRP2 (US Biologicals) were seeded onto the matrixand returned to 37° C., 5% CO₂ for 8 hours. Images were acquired usingthe Nikon Eclipse TS100 microscope at 4× magnification with a NikonCoolPix 995 digital camera. Results were quantified by counting thenumber of branch points. Endothelial tube formation was induced by SFRP2in a concentration-dependent manner at 8 hours (p=0.0006 at 7 nM) (FIG.8).

MATRIGEL Plug Assay:

The ability of mouse recombinant SFRP2 would stimulate angiogenesis in amouse MATRIGEL plug angiogenesis assay was evaluated. Female C57BL/6mice (8 weeks old) were injected s.c. with 0.5 ml of growth factorreduced basement membrane matrix (MATRIGEL) containing either mouserecombinant SFRP2 (800 ng/ml) with 30 U/ml heparin or PBS with 30 U/mlheparin for negative control. Seven days later the mice were sacrificedand the MATRIGEL plugs removed and evaluated for angiogenesis byhemoglobin concentration with the Drabkin's reagent. Evaluation of theangiogenic response by measurement of hemoglobin content showed a 3.3fold increase in SFRP2 plugs compared with the vehicle control (n=25SFRP2 plugs, n=26 control plugs, p=0.01, FIG. 9).

Endothelial Cell Apoptosis Assay:

Human coronary artery endothelial cells (HCAEC) were used for apoptosisassays because apoptosis could not be induced in the MEC cells. HCAECswere grown in 10-cm dishes (Becton Dickinson, Franklin Lakes, N.J.) withendothelial cell basal medium-2 (EGM-2) BulletKit media (Clonetics, SanDiego, Calif.) until 80% confluent. Medium was then replaced withoptimal medium according to different assays. The hypoxic condition wascreated by incubating HCAECs in EGM-2 media without BulletKit growthfactors at 37° C. in a hypoxia chamber with an atmosphere of 5% CO₂/95%N₂. The oxygen level in the chamber was controlled to 1.0%. Apoptosiswas determined by measuring the activity of cleaved caspase 3 by using acaspase-specific fluorogenic substrate according to the protocol for theCaspase 3 Assay Kit (Sigma). HCAECs were lysed after treatment withconcentrations of SFRP2 (70 pM and 700 pM) for 36 h under hypoxia. Then,5 μl of cell extract was incubated in reaction buffer at roomtemperature for 1 h. The enzyme-catalyzed release of 7-amino-4-methylcoumarin (AMC) was measured by a fluorescence microplate reader. It wasfound that SFRP2 protected against hypoxia induced endothelial cellapoptosis (p<0.02) (FIG. 10).

Gene Expression Analyses:

The downstream effects of SFRP2 on gene expression profiles wereevaluated using oligonucleotide microarrays. MEC cells were culturedwith and without SFRP2 700 pM for 16 hours. RNA was extracted andpurified using the Qiagen RNEASY Kit (Qiagen). The concentration andpurity of the total RNA was determined spectrophotometrically, andintegrity was verified using the RNA 6000 Nano LabChip (AgilentTechnologies) and Agilent 2100 bioanalyzer (Agilent Technologies).Biologic replicates were (n=3) for each group to improve confidence forthe average experimental-to-control intensity ratio for each gene. RNAfrom cells was labeled with Cy5-CTP using the Low-Input Linear RNAAmplification System (Agilent), and hybridized with equimolarconcentrations of Cy3-labeled mouse common reference RNA. Microarrayhybridizations were performed using Agilent Mouse Whole Genome 44 Koligonucleotide microarrays. After hybridization, the arrays werescanned on an Axon Gene Pix 4000b scanner (Axon Instruments, Inc.,Foster City, Calif.). The images were analyzed using Feature ExtractionV 9.1 software (Agilent). Gene expression values were quantified by theLog₂ ratio of red channel intensity versus green channel intensity(sample vs. reference), followed by loess normalization to remove theintensity dependent dye bias and variation. Data filtering andpre-processing were performed using custom Peri scripts. Data associatedwith this study are available at genome.unc.edulpubsup/breastTumor.Significantly differentially expressed genes were identified using anheteroscedastic (two-tailed, type 3) T test (p<0.01), and subsequentselection for an absolute mean fold change of >1.3. To interpret thegene lists and convert them into biological themes, the GATHER webinterface (Gene Annotation Tool to Help Explain Relationships;gather.genome.duke.edu) was used for analysis. Using this technique, 33differentially expressed mRNAs were found (FIG. 11).

Effect of SFRP2 on Wnt Pathways:

SFRP2 has been described as both a Wnt antagonist and agonist, but itseffects on Wnt signaling in endothelial cells have not been elucidated.To determine if SFRP2 mediates canonical Wnt signaling in endothelialcells, cytoplasmic and nuclear β-catenin levels were measured inSFRP2-treated endothelial cells. Mouse endothelial cells were plated in12-well plates and allowed to attach overnight. The next day, the mediawas changed and added to the wells with and without SFRP2 (700 pM).Cells were incubated for 16 hours, and the nuclear and cytoplasmicproteins were extracted by using NE-PER™ nuclear and cytoplasmicextraction reagent from PIERCE (Pierce Biotechnology) as described inthe manufacturer's manual. Western blot analysis was performed usingstandard methods, with primary antibody to the dephosphorylated (active)β-catenin antibody (Santa Cruz, Clone BDI480, catalog # sc-59893). Therewas no change in nuclear β-catenin in the SFRP2-stimulated cells,suggesting that the angiogenic property of SFRP2 is not mediated throughthe canonical Wnt signaling pathway (FIG. 12).

To evaluate the role of the non-canonical Wnt/Ca⁺⁺ pathway in SFRP2induced angiogenesis, nuclear dephosphorylated NFATc3 protein levelswere compared in control and SFRP2-treated endothelial cells. MEC cellswere plated in 12-well plates and allowed to attach overnight. The nextday, the media was changed and added to the wells with and without SFRP2(700 pM). Cells were incubated for 1, 2, 4, 8 and 16 hours, and thenuclear proteins were extracted by using NE-PER™ nuclear and cytoplasmicextraction reagent from PIERCE (Pierce Biotechnology) as described inthe manufacturer's manual. The Western blot analysis was performed usingstandard methods, with primary antibody to the dephosphorylated (active)β-catenin antibody or NFATc3. As above, there was no change in nuclearβ-catenin in the SFRP2-stimulated endothelial cells (p=0.4, FIG. 13),suggesting that the angiogenic property of SFRP2 is not mediated throughthe canonical Wnt signaling pathway. It was found that NFATc3 wasincreased at 30 minutes in the nuclear fraction of SFRP2-treatedendothelial cells (FIG. 13).

To evaluate whether tacrolimus inhibits SFRP2 induced tube formation,MEC cells were treated as above with SFRP2 7 nM with and withouttacrolimus 100 uM for 8 hours and branch points were determined asdescribed above. To evaluate whether tacrolimus reversed establishedtubes, cells were incubated with SFRP2 7 nM for 8 hours and then BSA ortacrolimus 100 μM was added to SFRP2 treated cells for an additional 4hours, and then the number of branch points were counted. Tacrolimusinhibited SFRP2 induced tube formation in MEC cells (FIG. 14).Tacrolimus was not cytotoxic to MEC cells, as only 5% oftacrolimus-treated cells took up trypan blue dye (data not shown). Inaddition, after tubes were formed with treatment with SFRP2, tacrolimusreversed SFRP2-induced tube formation (FIG. 15).

A Mouse Model of Angiosarcoma Overexpresses SFRP2 Protein:

In order to study whether inhibition of SFRP2 will inhibit tumor growth,we set out to identify a tumor model that overexpresses SFRP2. To dothis, a transformed mouse endothelial cells line were studied. Ms1 cellswere generated by immortalizing murine endothelial cells by expressingthe temperature-sensitive large T antigen (gift of Dr. Jack Arbiser,Emory University). Upon implantation into mice, these cells form dormanthemangiomas. Ms1 cells were then transfected with Ras (SVR cell line),and this cell line forms angiosarcomas when injected into nude mice.Protein lysates were collected from MS1 and SVR cell lines and, usingWestern blot analyses probing for SFRP2, found that SFRP2 was increasedin SVR cells (FIG. 16). Since this cell line forms aggressiveangiosarcomas, it is an ideal mouse model to study inhibition of tumorgrowth by inhibitors of SFRP2.

Tube Formation Assay:

The tube formation properties of SFRP2 on MEC cells were evaluated usingan endothelial cell tube formation assay. ECMATRIX (Chemicon) wasthawed, diluted and solidified in a 96 well plate according to themanufactures instructions. 1×10⁴ cells/well in 150 μl of DMEM (cellgro)with 10% FBS (HyClone) and a concentration range (3-3000 pM) of SFRP2(US Biologicals) were seeded onto the matrix and returned to 37° C., 5%CO₂ for 8 hours. Images were acquired using the Nikon Eclipse TS100microscope at 4× magnification with a Nikon CoolPix 995 digital camera.Results were quantified by counting the number of branch point. Toevaluate whether tacrolimus inhibits SFRP2 induced tube formation, MECcells were treated as above with SFRP2 30 nM with and without tacrolimus(1 μM-100 uM) for 8 hours and branch points were determined as describedabove. To evaluate whether inhibitors of SFRP2-mediated angiogenesiswould inhibit the growth of SVR tumor cells, SVR cells were treated withtacrolimus (1 μM-100 μM) or with a rabbit polyclonal antibody to SFRP-2(H-140) (Santa Cruz Biotechnology, Santa Cruz, Calif., catalogue 4sc-13940) in the tube formation assay.

As shown above, MEC endothelial tube formation was induced by SFRP2 in aconcentration-dependent manner at 8 hours (p=0.0006 at 7 nM) (FIG. 17A).To further evaluate whether the angiogenic effects of SFRP2 weremediated through NFAT, endothelial cells were treated in a tubeformation assay with SFRP2 (30 nM) with and without the calcineurininhibitor tacrolimus. Tacrolimus (1 μM) inhibited SFRP2 induced tubeformation by 64%±(0.002) (FIG. 17B). Tacrolimus was not cytotoxic to MECcells, as only 5% of tacrolimus-treated cells took up trypan blue dye(data not shown). Tube formation in SVR angiosarcoma cells were alsoinhibited by tacrolimus (FIG. 17C), and SVR tube formation was inhibitedby a polyclonal antibody to SFRP2, suggesting that SFRP2 is required fortube formation in this angiosarcoma tumor cell line (FIG. 17D).

The ability for tacrolimus to inhibit SFRP2 and VEGF stimulated 2H11endothelial tube formation in vitro was studied in a MATRIGEL tubeformation assay. Tacrolimus inhibited endothelial tube formation in bothSFRP2 (FIG. 18) and VEGF (FIG. 19) stimulated endothelial cells.

As a further test of the contribution of SFRP2 to endothelial tubeformation, it was evaluated whether loss of function of SFRP2 wouldinhibit SVR angiosarcoma tube formation. This was studied two differentways, first with a blocking antibody to SFRP2, and then using siRNA toSFRP2. SVR cells were plated in MATRIGEL in a tube formation assay andtreated with a polyclonal antibody to SFRP2. SYR tube formation wasinhibited with the polyclonal antibody to SFRP2 in a concentrationdependent manner (FIGS. 20A-B). Next, SVR cells were transfected withsiRNA to SFRP2 from Santa Cruz and sham control. SVR cells weretransfected with 72 μM siRNA for SFRP2 (FRP-2 siRNA (sc-40001, SantaCruz Biotechnology) is a pool of 3 target-specific 20-25 nt siRNAsdesigned to knock down SFRP2 gene expression). The three sequences are5′-GAGAUAACGUACAUCAACA-3′ (SEQ ID NO:10), 5′-CAAGCUGCAAUGCUAGUUU-3′ (SEQID NO:11), 5′-CCAUGUCAGGCGAAUUGUU-3′(SEQ ID NO:12). The control siRNA(sc-36869, Santa Cruz Biotechnology) that was used contains a scrambledsequence that does not lead to the specific degradation of any knowncellular mRNA. SVR cells were maintained in Dulbecco's modified Eagle'smedium supplemented with 10% fetal calf serum. After 72 h oftransfection using LIPOFECTAMINE™ RNAiMAX Transfection reagent(Invitrogen) according to the manufacturer's protocol, cells wereharvested and ready for Western blot analyses and tube formation assay.Cells were seeded for a 4 hour tube formation assay. siRNA to SFRP2transfected cells had a 70% reduction in tube formation compared to shamtransfected cells (FIG. 21). These studies demonstrate that SFRP2 isrequired for angiosarcoma tube formation.

SFRP2 belongs to a large family of secreted frizzle-related proteins(SFRPs) which are related to the Wnt-signaling cascade. This proteincontains a cysteine-rich domain which is homologous to the putativeWnt-binding domain. The Wnt-signaling network influences biologicalprocesses ranging from developmental cell fate to cell adhesion andapoptosis. Recent data suggests that the Wnt signaling pathway isinvolved in formation and remodeling of blood vessels.

Wnt proteins have been grouped into two classes—canonical andnoncanonical. Canonical Wnts stabilize β-catenin, thereby activatingtranscription of Tcf/LEF target genes. SFRP2 has been reported to be anantagonist of the canonical Wnt signaling pathway by binding directly toWnts, thereby altering their ability to bind to the Wnt receptorcomplex. However, in our study we found no change in cytoplasmic ornuclear β-catenin levels in SFRP2-treated endothelial cells at theconcentration and time points which induced tube formation andmigration, indicating that SFRP2 is not mediating angiogenesis via thecanonical-Wnt signaling pathway.

Noncanonical Wnts activate other signaling pathways, such as theWnt/Ca²⁺ pathway which regulate NFATc. The NFAT family consists of fourmembers (NFATc1-c4), which exist as transcriptionally inactive,cytosolic phosphoproteins. NFAT nuclear localization is dependent on adynamic import-export balance between the activity of theCa²⁺/calmodulin-dependent phosphatase, calcineurin, and the activity ofserine/threonine kinases. Loss-of-function mutants have shown that NFATsignaling is crucial for normal heart valves and vascular developmentduring embryogenesis. Postnatally, this pathway contributes to theregulation of cell growth, differentiation, and cell cycle progressionin various cell types, and there is increasing data supporting acritical role of NFAT in mediating angiogenic responses.

Wnt5a has been shown to be a mediator of the non-canonical Wnt pathway,and SFRP2 has previously been shown to bind to Wnt5a in the nanomolarrange. Based on this, it was evaluated whether SFRP2 activated thenon-canonical Wnt pathway in endothelial cells. Tacrolimus, acalcineurin inhibitor, inhibited SFRP2 induced tube formation,suggesting that SFRP2 induces tube formation via the non-canonicalWnt-Ca²⁺ signaling pathway, resulting in nuclear translocation of NFATc.

Example 5 Antibodies to SFRP2

An analysis of the amino acid sequence of the human SFRP2 sequence wasperformed to determine candidate epitopes for making synthetic peptidesfor injection into animals to develop monoclonal antibodies to humanSFRP2. Seven candidate sequences were identified based on theirpredicted immunogenicity: AA29-40: GQPDFSYRSNC (SEQ ID NO:1); AA85-96:KQCHPDTKKELC (SEQ ID NO:2); AA119-125 VQVKDRC (SEQ ID NO:3); AA138-152:DMLECDRFPQDNDLC (SEQ ID NO:4); AA173-190: EACKNKNDDDNDIMETLC (SEQ IDNO:5); AA202-220 EITYINRDTKIILETKSKT-Cys (SEQ ID NO:6); AA270-295: ITSVKRWQKGQREFKRISRSIRKLQC (SEQ ID NO:7). Mice were immunized against thefirst five of the above peptide sequences with a second round ofimmunization one month later and bleeds performed two weeks later. AnELISA was performed to determine the titer of the mice to the peptides,which demonstrated that the mice responded to the various immunogens.Sera from the mouse immunized against the immunogen used as the epitopecorresponding to amino acids AA202-AA220 (which was designated AbB) andAA270-AA295 (which was designated AbC) inhibited SVR tube formationcompared to control mouse sera (FIG. 22), indicating that these peptidesequence are functionally active. These two peptides were selected forthe production of a monoclonal antibody.

Monoclonal antibodies for the epitope corresponding to AbB (AA202-AA220)were prepared. A tertiary injection of antigen had be performed and themice were boosted by a single intraperitoneal injection. The titers tothe immunogen in the injected mice were elevated, and two mice wereselected for spleen harvest. The mice selected underwent a final i.p.immunization three weeks after the last immunization. These mice weresacrificed 3 days after the final boost and blood was collected. Thespleen was removed and fused with myeloma cells for hybridoma formation.The fusion of the spleen cells was with P3X63-Ag8.653 (ATCC CRL-1580)cells using a 50% PEG solution. The fusion was plated in 96 well platesat a total cell concentration of −1.5×10⁵ cells per well in the FIATselection media. The fusion plates were fed after 7 days with HAT media,Fusion plates were screened 14 days after the fusion was performed. Thescreening was performed by plating SVR angiosarcoma cells in a MATRIGELtube formation assay in 96 well plates. The angiosarcoma cells weresuspended in 150 μl of antibody containing growth media for 90 differentsamples and control media, After 6 hours, pictures were taken and thenumber of branch points for each well was counted. The controls formed39 branchpoints. There were 61 samples that had a >50% inhibition oftube formation. Eight samples that each had less than 4 branchpoints(FIG. 23) were selected to use for further subcloning, and 32 samplesthat have >50% inhibition were frozen back.

Example 6 SFRP2 as a Biomarker for Breast Cancers

To investigate whether SFRP2 is a biomarker for breast cancer, thepresence of SFRP2 protein in serum from patients with breast cancercompared to normal control was tested. Serum was obtained from the UNCtissue procurement facility, where serum was collected under an IRBapproved protocol. Patient serum was diluted 1:14 and filtered. Thetotal protein level was measured using the Bio-Rad Protein assay. Equalamount of protein was loaded and Western blot was performed according tothe standard method. The blot was probed with the SFRP2 antibody. It wasfound that SFRP2 is present in control and breast cancer patient serumbut more highly expressed in the latter (p<0.0001, FIG. 24).

Example 7 SFRP2 is a Broad Spectrum Vascular Target

Using IHC with antibodies to SFRP2 on paraffin-embedded human tumors,the vascular expression of SFRP2 in angiosarcoma, colon cancer, prostatecancer, lung cancer, ovarian cancer, hepatocellular carcinoma, renalcell carcinoma, and pancreatic cancer was evaluated. It was found thatSFRP2 is strongly expressed in all tumors, making SFRP2 a broad spectrumvascular target (FIG. 25).

Example 8 Tacrolimus Inhibits Angiogenesis In Vivo

To test the ability of tacrolimus to inhibit angiogenesis in vivo, SVRmouse angiosarcoma cells (0.5×10⁶) were injected into the flank of6-week-old nude male mice obtained from Charles River BreedingLaboratories. Treatment was initiated on the day after inoculation. Micereceived 3 mg/kg/daily tacrolimus or vehicle control suspended in 20%Intralipid (Baxter Healthcare, Deerfield, Ill.) in a total volume of 0.3ml intraperitoneally (i.p.), and were treated daily for 19 days. Serialcaliper measurements of perpendicular diameters were used to calculatetumor volume using the following formula: (shortest diameter)²×(longestdiameter)×0.52. Differences in tumor volume over time were analyzed witha two way ANOVA. A P value of ≦0.05 indicated a statisticallysignificant reduction in tumor growth of the treated group compared tothe control group. Treatment with tacrolimus (n=14) for 19 days waseffective at suppressing the growth of SVR angiosarcoma tumor in nudemice as compared with control (n=14). Treatment with tacrolimus reducedmean tumor volume by 46% at day 19 (589±129 mm³ vs 315±93 mm³, two-wayANOVA, p=0.04, FIG. 26). There were no signs of toxicity (i.e., nodiarrhea, infection, lethargy, or weight loss) after 19 days oftreatment.

In a second study, MMTV-neu transgenic mice were treated with tacrolimus3 mg/kg/daily or no treatment. Treatment began when tumors were palpableand continued for 21 days. Tumor volumes were monitored with serialultrasounds. A two-tailed T-test was used to determine the differencebetween growth rates of the tumors between treated and non-treatedtumors. The groups were significantly different (P=0.04, two-tailedt-test) at the end of the study on day 21, with a 59% reduction ingrowth rate (FIG. 27). There were no signs of toxicity (i.e., diarrhea,infection, lethargy, or weight loss) after treatment.

Example 9 Angiogenic Function of Jak3

Cell Culture:

Human coronary artery cells (HCAEC) were cultured in endothelial cellbasal medium-2 (EGM-2) with BulletKit growth supplements (Clonetics, SanDiego, Calif.). Cells were passaged at 80-90% confluence using trypsinwithout EDTA (Invitrogen, Carlsbad, Calif.). Passages 3-9 were used inexperiments.

Chick Chorioallantoic Membrane (CAM) Assay:

Fertilized chickens eggs (NC State University Chicken Research Faint)were incubated at 100.4° F. in an egg turner (Model#1588 GenesisHova-Bator, GQF Mfg. Co., Inc.) for 3 days. On day 3, the eggs werecracked into sterile 100×25 mm dishes and incubated at 99° F., 5% CO₂,65% humidity for 5 days. For application of drug onto the CAM, Whatmangrade 1 filter paper was cut into circles with a 6 mm diameter paperpunch and autoclaved. To decrease inflammatory effects of the disk, theywere soaked in 1 ml of 3.0 mg/ml cortisone acetate in absolute ETOH andair dried for 60 min in a laminar flow hood under ultraviolet light. Onday 8, disks were inoculated with 7 μl 0.1% BSA in PBS for control CAMsor 100 ng Jak3/7 μl, 0.1% BSA in PBS for Jak3-treated CAMs (n=16 controldisks and n=16 Jak3-treated disks). Disks were then placed on the outerthird of the CAM, 2-3 mm from a major vessel. CAMs were evaluated undera stereomicroscope on day 3 after disk placement. Pictures were takenwith a Wild M-4 70 Macrosystem and angiogenesis was quantified usingMetamorph Software with an angiogenesis module. After 3 days, Jak3induced angiogenesis on the CAM with a statistically significantincrease in number of branch points (0.0001), segments (0.0001), tubepercent area covered (0.0001), and total tube length (0.0001) (FIG. 28).

Scratch Wound Assay:

HCAEC were plated at 10,000 cells/well onto a 96 well plate and allowedto become confluent in EGM-2 with BulletKit growth supplements. Thecells were quiesced in EGM-2 with 0.1% FBS without BulletKit growthsupplements for 18 hours. The wound was formed using a 1 ml pipette tip.A 20 nM-200 pM dose curve of recombinant human Jak3 (Millipore,Temecula, Calif.) was added to the cells. Each concentration wasperformed in triplicate and the experiment was repeated three times withsimilar results. Migration distance was measured at 12 hours and thenevery 4 hours until wound closure in all wells. Data were recorded aspercent of wound closed at each time point. Jak3 increased endothelialcell migration in the nanomolar concentration (p<0.03 at 20 hours,p<0.001 at 28 hours) (FIG. 29).

Tube Formation Assay:

ECMATRIX (Chemicon, Temecula, Calif.) was thawed, diluted, andsolidified in a 96 well plate according to the manufacturer'sinstructions. HCAEC were seeded onto the matrix at 2,000 cells/well in150 μl of EGM-2 with 5% FBS without BulletKit growth supplements. A 20nM-200 pM dose curve of recombinant human Jak3 was added to the cellsand the plates were returned to 37° C., 5% CO₂ for 8 hours. Eachconcentration was performed in triplicate and the experiment wasrepeated three times with similar results. Wells were evaluated under aNikon Eclipse TS100 microscope at 4× magnification and pictures weretaken with a Nikon CoolPix 995 digital camera. Angiogenesis wasquantified by counting branch points in the resulting image. Endothelialtube formation was induced by Jak3 in a concentration-dependent mannerat 8 hours (p=0.04 at 200 pM, p=0.0001 at 20 nM) (FIG. 30).

Endothelial Cell Apoptosis Assay:

HCAEC were seeded onto a 96 well plate at 2,000 cells/well in EGM-2 withBulletKit growth supplements. Cells were grown for 18 hours and mediawas replaced with EGM-2 without BulletKit growth supplements and a 20nM-200 pM dose curve of recombinant human Jak3 was added to the cells.The plate was incubated in hypoxic conditions (37° C. in a hypoxiachamber with an atmosphere of 5% CO₂/95% N₂ with an oxygen level of1.0%) for 36 hours. Apoptosis was determined by measuring the activityof cleaved caspase 3 by using a caspase-specific fluorogenic substrateaccording to the protocol for the Apo-ONE® Homogeneous Caspase-3/7 Assay(Promega, Madison, Wis.). Briefly, control and treated HCAEC were lysedin 100 μL of Apo-ONE® Caspase-3/7 reagent and incubated in that reagentat room temperature for 1 h. The caspase 3 activation of theprofluorescent substrate rhodamine 110,bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide(Z-DEVD-R110) was measured by a fluorescence microplate reader. It wasfound that Jak3 protected against hypoxia induced endothelial cellapoptosis (p<0.05) (FIG. 31).

Endothelial Cell Proliferation Assay:

HCAEC were seeded on a 96 well plate at a concentration of 2000cells/well and allowed to proliferate for 24 hrs in EGM-2 with BulletKitgrowth supplements. The cells were then quiesced in EGM-2 with 0.1% FBSwithout BulletKit growth supplements for 18 hours. Media was replacedwith fresh EGM-2 with BulletKit growth supplements and the cells weretreated in triplicate with: PBS alone; recombinant murine VEGF (60ng/mL); or Jak3 (at a concentration of 200 nM-200 pM). After 48 h, 10 μlof the colorimetric compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to each well and allowedto incubate for 4 h at 37° C. All but 25 μl of media in each well wasremoved and 50 μl dimethyl sulfoxide (DMSO) was added. Following a 10min incubation at 37° C., the A540 was measured using a microplatereader. A540 was converted to number of cells based on a standard curvedcreated by seeding a 96-well plate with known concentrations of cells,as determined by hemocytometry, and measuring their A540 after 4 hrsincubation with MTT. Jak3 increased cell proliferation at 48 hours(p=0.007 at 20 nM) (FIG. 32).

Effect of STAT3 on Tube Formation:

The role of STAT3 activation in Jak3-mediated tube formation wasevaluated using a small peptide inhibitor of phosphorylated STAT3(P-STAT3). ECMATRIX was solidified in wells of a 96 well plate. HCAECwere seeded onto the matrix at 2,000 cells/well in 150 μl of EGM-2 with5% FBS without additional BulletKit growth supplements. Wells weretreated in triplicate with: PBS alone, PBS+100 μM P-STAT3 inhibitor, 20nM Jak3, or 20 nM Jak3+100 μM P-STAT3 inhibitor. Wells were photographedat 8 hours and tube formation was quantified by counting branch pointsin the resulting image. Addition of STAT3 inhibitor preventedJak3-mediated tube formation, indicating that the Jak3 signal ismediated through the STAT3 pathway (FIG. 33).

Example 10 SFRP2 Antibody Inhibits Tumor Growth In Vivo

The ability of a monoclonal antibody recognizing SFRP2 to inhibit tumorgrowth in vivo was tested in a nude mouse assay. The SFRP2 MAb subclone80.8.6 was first tested with SVR angiosarcoma xenografts. Six week oldnude mice were injected with 1×10⁶ SVR angiosarcoma cells. The day afterinjections treated mice received SFRP2 MAb(80.8.6) 4 mg/kg i.v. twiceweekly (n=5), and controls received equal volumes of buffer. At day 14there was a 65% decrease in tumor volume (*p=0.03, two-tailed student'st-test) (FIG. 34). No toxicity or weight loss was seen in the mice.

The SFRP2 MAb subclone 80.8.6 was next tested with MDA-MB-231 breastcancer xenografts. Six week old female nude mice were injected with1×10⁶ MDA-MB-231 cells. After 4 weeks, when tumors were palpable, micereceived SFRP2 MAb (80.8.6) 4 mg/kg i.v. twice weekly (n=12), andcontrols received equal volumes of buffer. At day 8 the median tumorvolume for controls was 1200 mm³, and for SFRP2 MAb treated mice was 580mm³, which represents a 52% decrease in tumor volume (p=0.04, two-tailedstudent's t-test). No toxicity or weight loss was seen in the mice.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. An isolated monoclonal antibody or anantigen binding fragment thereof which specifically binds an epitopecomprised of amino acids selected from the group consisting of aminoacids EITYINRDTKIILETKSKT-Cys (SEQ ID NO:6) andITSVKRWQKGQREFKRISRSIRKLQC (SEQ ID NO:7).
 2. The monoclonal antibody oran antigen binding fragment thereof of claim 1, which specificallyrecognizes an epitope which is amino acids EITYINRDTKIILETKSKT-Cys (SEQID NO:6).
 3. The monoclonal antibody or an antigen binding fragmentthereof of claim 1, wherein the monoclonal antibody is a recombinantprotein.
 4. The monoclonal antibody or an antigen binding fragmentthereof of claim 3, which is a single chain monoclonal antibody.
 5. Themonoclonal antibody or an antigen binding fragment thereof of claim 1,wherein the fragment is an Fab, Fab′, F(ab′)₍₂₎, or Fv fragment.
 6. Themonoclonal antibody or an antigen binding fragment thereof of claim 1,which is a chimeric antibody.
 7. The monoclonal antibody or an antigenbinding fragment thereof of claim 1, which is a humanized antibody.
 8. Acomposition comprising the monoclonal antibody or an antigen bindingfragment thereof of claim 1 and a pharmaceutically-acceptable carrier.9. A composition comprising the monoclonal antibody or an antigenbinding fragment thereof of claim 5 and a pharmaceutically-acceptablecarrier.